LPA Receptor Agonists and Antagonists and Methods of Use

ABSTRACT

Disclosed are compounds according to formula (I) as well as pharmaceutical compositions which include those compounds. Also disclosed are methods of using such compounds, which have activity as agonists or as antagonists of LPA receptors; such methods including inhibiting LPA activity on an LPA receptor, modulating LPA receptor activity, treating cancer, enhancing cell proliferation, treating a wound, treating apoptosis or preserving or restoring function in a cell, tissue, or organ, culturing cells, preserving organ or tissue function, and treating a dermatological condition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of and claims thepriority benefit of U.S. patent application Ser. No. 10/963,085, filedOct. 12, 2004, which claimed the benefit of priority of earlier-filedProvisional Patent Application Ser. No. 60/509,971, filed Oct. 9, 2003.The disclosures of both applications are incorporated herein byreference in their entirety.

STATEMENT OF GOVERNMENT RIGHTS

This invention was funded, in part, by the National Institutes of HealthGrant No. CA92160. The U.S. government may have certain rights in thisinvention.

FIELD OF THE INVENTION

This invention relates to lysophosphatidic acid (“LPA”) derivativeswhich have activity as either agonists or antagonists on LPA receptorsand various therapeutic uses thereof including, but not limited to,prostate cancer therapy, ovarian cancer therapy, and wound healing.

BACKGROUND OF THE INVENTION

Non-transformed cells require growth factors for their survival andproliferation. In addition to polypeptide growth factors, an emergingclass of lipids with growth factor-like properties has been discovered,collectively known as phospholipid growth factors (PLGFs). Although theyhave similar pharmacologic properties in inducing the proliferation ofmost quiescent cells (Jalink et al., 1994a; Tokumura, 1995; Moolenaar etal., 1997), PLGFs can be sub-divided structurally into two broadcategories. The first category contains the glycerophospholipidmediators (GPMs), which possess a glycerol backbone. Exemplary GPMsinclude LPA, phosphatidic acid (PA), cyclic phosphatidic acid(cyclic-PA), alkenyl glycerol phosphate (alkenyl-GP), andlysophosphatidyl serine (LPS). The second category contains thesphingolipid mediators (SPMs), which possess a sphingoid base motif.Exemplary SPMs include sphingosine-1-phosphate (SPP),dihydrosphingosine-1-phosphate, sphingosylphosphorylcholine (SPC), andsphingosine (SPH).

LPA (Tigyi et al., 1991; Tigyi and Miledi, 1992), PA (Myher et al.,1989), alkenyl-GP (Liliom et al., 1998), cyclic-PA (Kobayashi et al.,1999), SPP (Yatomi et al., 1995), and SPC (Tigyi et al., 2000) have beendetected in serum. These lipid mediators have been identified andcharacterized. There are still yet unknown PLGFs present in the serumand plasma that exhibit growth factor-like properties (Tigyi and Miledi,1992). LPA, at a concentration of ≈20 μM, is the most abundant PLGFpresent in the serum (Tigyi and Miledi, 1992; Jalink et al., 1993).

In eukaryotic cells, LPA is a key intermediate in the early stages ofphospholipid biosynthesis, which takes place predominantly in themembrane of endoplasmic reticulum (ER) (Bosch, 1974; Bishop and Bell,1988). In the ER, LPA is derived from the action of Acyl-CoA onglycerol-3-phosphate, which is further acylated to yield PA. Because therate of acylation of LPA to PA is very high, very little LPA accumulatesat the site of biosynthesis (Bosch, 1974). Since LPA is restricted tothe ER, its role as a metabolic intermediate is most probably unrelatedto its role as a signaling molecule.

LPA is a constituent of serum and its levels are in the low micromolar(μM) range (Eicholtz et al., 1993). This level is expected because LPAis released by activated platelets during the coagulation process.Unlike serum, it is not detectable in fresh blood or plasma (Tigyi andMiledi, 1992; Eicholtz et al., 1993). LPA that is present in the serumis bound to albumin, and is responsible for a majority of theheat-stable, and non-dialysable biological activity of the whole serum(Moolenaar, 1994). The active serum component that is responsible foreliciting an inward chloride current in Xenopus oocyte was identified tobe LPA (18:0) (Tigyi and Miledi, 1992). The bulk of the albumin-boundLPA (18:0) is produced during the coagulation process, rather than bythe action of lysophospholipase D (PLD) on lyso-PC. The latter pathwayis responsible for the presence of LPA in ‘aged’ plasma that has beende-coagulated by the action of heparin or citrate plus dextrose(Tokumura et al., 1986). Furthermore, LPA is not present in plasma thathas been treated with EDTA. This fact implies that plasmalysophospholipase may be Ca²+-dependent (Tokumura et al., 1986).

The role of albumin is to protect LPA from the actions of phospholipasespresent in the serum (Tigyi and Miledi, 1992). Tigyi and Miledisuggested that albumin not only acts as a carrier of LPA in the bloodstream, but also increases its physiological half-life. There are yetunidentified lipid mediators present in serum albumin that mimic theactions of LPA in eliciting chloride current in Xenopus oocytes.

LPA-responsive cell types extend from those of slime mold amoebae andXenopus oocyte to mammalian somatic cells. Thus, it seems likely thatthe source of LPA and its release may not be restricted only toactivated platelets. Recent experiments showed that, on stimulation bypeptide growth factors, mammalian fibroblasts rapidly produce LPA, whichis followed by its release into the extracellular medium (Fukami andTakenawa, 1992).

There is evidence that relatively high amounts of bioactive LPA ofunknown cellular origin are present in the ascitic fluid of ovariancancer patients (Xu et al., 1995a), and that the ascitic fluid from suchpatients is known to possess potent mitogenic activity for ovariancarcinoma cells (Mills et al., 1988; Mills et al., 1990). It remains tobe established whether it is secreted by tumor cells into theextracellular fluid, secreted by leukocytes, or produced from morecomplex lipids via the actions of various phospholipases.

GPMs and SPMs elicit a wide variety of cellular responses that span thephylogenetic tree (Jalink et al., 1993a). LPA induces transient Ca²⁺signals that originate from intracellular stores in a variety of cellssuch as neuronal (Jalink et al., 1993; Durieux et al., 1992), platelets,normal as well as transformed fibroblasts (Jalink et al., 1990),epithelial cells (van Corven et al., 1989; Moolenaar, 1991), and Xenopusoocytes (Tigyi and Miledi, 1992; Durieux et al., 1992; Fernhout et al.,1992). LPA induces platelet aggregation (Schumacher et al., 1979;Tokumura et al., 1981; Gerrard et al., 1979; Simon et al., 1982) andsmooth muscle contraction (Tokumura et al., 1980; Tokumura et al.,1994), and upon intravenous administration it induces species-dependentchanges in blood pressure ((Schumacher et al., 1979; Tokumura et al.,1978).

LPA, when added to quiescent fibroblasts, stimulates DNA synthesis andcell division (van Corven et al., 1989; van Corven et al., 1992). Thegrowth-like effects of LPA do not require the presence of peptide growthfactors. This observation makes LPA different from endothelin orvasopressin, which require the presence of insulin or epidermal growthfactor (Moolenaar, 1991) to sustain cell proliferation. Notably, in Sp2myeloma cells, LPA demonstrated an antimitogenic response, which wasmediated by an increase in cAMP levels (Tigyi et al., 1994; Fischer etal., 1998). Unlike the mitogenic pathway, the anti-mitogenic pathway wasnot affected by pertussis toxin (PTX). Also, on addition of forskolinand isobutyl methyl xanthin, the antimitogenic actions of LPA in Spmyeloma cells were additive (Tigyi et al., 1994). In various cell types,LPA causes cytoskeletal changes, which include formation of focaladhesions and stress fibers in fibroblasts (Ridley and Hall, 1992). LPAalso promotes the reversal and suppression of neuroblastomadifferentiation by inducing the retraction of developing neurites(Jalink et al., 1994a; Jalink et al., 1994b). Addition of nanomolar(nmol) amounts of LPA (Jalink and Moolenaar, 1992) to serum-starvedN1E-115 neuroblastoma cells caused immediate neurite retraction, whichwas accompanied by rapid, but transient, rounding of the cell body(Jalink et al., 1993b). When a continuous presence of LPA is provided,neuroblastoma cells maintain their undifferentiated phenotype, but failto undergo mitosis (Jalink et al., 1993b). Additional factors, such asinsulin-like growth factors, are required for the progression of thecell cycle. Once the cells have undergone morphological differentiation,the addition of LPA reverses this morphological change. Thus,LPA-induced neurite retractions result from the contraction of theactin-cytoskeleton, rather than from loss of adhesion to the substratum(Jalink et al., 1993b; Jalink et al., 1994b).

LPA, similar to other physiological chemoattractants (e.g.,interleukin-8), induces cell migration by a haptotactic mechanism inhuman monocytes (Zhou et al., 1995). In addition to inducing cellmigration, LPA promotes the invasion of hepatoma and carcinoma cellsinto the monolayer of mesothelial cells (Imamura et al., 1993). Themechanism that underlies this invasion is still unclear, but it may bedue to enhanced cell motility and increased cell adhesion. Finally, LPAis also known to block neonatal cardiomyocyte apoptosis (Umansky et al.,1997).

A unique natural phospholipid, namely cyclic-PA, was shown to beresponsible for cellular actions that were similar to or opposite toother GPMs, depending on the cell type. When tested on the Xenopusoocyte, it elicited chloride current just like other GPMs; but itsresponse was not desensitized by LPA (Fischer et al., 1998).Murakami-Murofushi et al. (1993) showed that cyclic-PA exhibitedantiproliferative actions, unlike LPA, which induces proliferation.

PLGF receptors (PLGFRs) belong to a seven-transmembrane (7 TM) guaninenucleotide-binding regulatory protein (G protein)-coupled receptors(GPCR) superfamily. Seven-TM GPCRs are a family of cell-surfacereceptors that mediate their cellular responses via interacting with theheterotrimeric G-protein. A number of LPA receptors have been identifiedincluding, among others, EDG-2, EDG-4, EDG-7, and PSP-24. A phylogenetictree illustrating the relatedness of these LPA receptors and others isshown in FIG. 1.

In 1996, Hecht et al. used differential hybridization to clone a cDNAencoding a putative serpentine receptor from mouse neocortical celllines (Hecht et al., 1996). The gene was termed as ventricular zonegene-1 (Vzg-1). The gene was expressed in cortical neurogenic regionsand encoded a protein with a molecular weight of 41 kDa (364 aminoacids). Vzg-1 was very similar to an unpublished sheep sequence termedendothelial differentiation gene-2 (EDG-2). The same cDNA was alsoisolated as an orphan receptor from mouse and bovine libraries, and wasknown as rec1.3 (Macrae et al., 1996). It was widely distributed in themouse tissue, with the highest expression in the brain and heart.

In 1996, Guo et al., using a PCR base protocol, isolated anotherputative LPA receptor PSP-24 (372 amino acids) from Xenopus oocyte (Guoet al., 1996). This receptor showed little similarity withVzg-1/EDG-2/rec1.3 (Guo et al., 1996). A sequence based search forsphingolipid receptors, using the cDNA sequence of the EDG-2 human LPAreceptor, led to two closely related GPCRs, namely, rat H218 (EDG-5, 354amino acids) and EDG-3 (378 amino acids) (An et al., 1997a). Northernanalysis showed a high expression of mRNA that encoded EDG-3 and EGD-5in heart tissue.

The recent identification of EDG-2 as a functional receptor for LPAprompted An et al. to perform a sequence-based search for a novelsubtype of LPA receptor (An et al., 1998a). A human cDNA, encoding aGPCR, was discovered and designated EDG-4 (An et al., 1998a). Northernblot analysis showed that, although EDG-2 and EDG-4 both serve as GPMreceptors, their tissue distributions were very different. Unlike EDG-2,EDG-4 was primarily expressed in peripheral blood leukocytes and testes(An et al., 1998a).

PCR amplification cDNA from human Jurkat T cells identified a previouslyunknown GPCR belonging to the EDG family. The identified GPCR wasdesignated EDG-7. It has a molecular mass of 40 kDa (353 amino acids).Northern blot analysis of EDG-7 expression in human tissues showed thatit is expressed in heart, pancreas, prostate, and testes (Bandoh et al.,1999). Thus, there are two distinct families of PLGF receptors PSP24 andEDG, with a total of ten individual PLGFRs (FIG. 1). The list continuesto grow.

These various receptors can be classified based on their ligandspecificities for GPMs or SPMs, as shown in Table 1 below. TABLE 1Phospholipid Growth Factor Receptor (PLGFR), Length and Principle LigandNumber of Principle PLGFR amino acids Ligand EDG-1 381 SPP EDG-2 364 LPAEDG-3 378 SPP EDG-4 382 LPA EDG-5 354 SPP EDG-6 385 SPP EDG-7 353 LPAEDG-8 400 SPP Xenopus PSP24 372 LPA Murine PSP24 373 LPA

Xenopus PSP24 and murine expressed PSP24 specifically transduceGPM-evoked oscillatory chloride-currents (LPA, Fischer et al., 1998).These are not structurally homologous to the EDG family (Tigyi andMiledi, 1992; Fernhout et al., 1992). The EDG family can be divided intotwo distinct subgroups. The first group includes EDG-2, EDG-4, andEDG-7, which serve as receptors for only GPM (Hecht et al., 1996; An etal., 1998a; Bandoh et al., 1999; An et al., 1998b) and transmit numeroussignals in response to ligand binding. The second group involves EDG-1,EDG-3, EDG-5, EDG-6, and EDG-8, and is specific for SPMs (An et al.,1997a; Im et al., 2000; van Brocklyn et al., 1998; van Brocklyn et al.,2000; Spiegel and Milstein, 2000). Principle tissue expression of thevarious PLGFR's is shown in Table 2 below. TABLE 2 Human TissueExpression of Phospholipid Growth Factor Receptors PLGFR Human Tissuewith Highest Expression EDG-1 Ubiquitous EDG-2 Cardiovascular, CNS,Gonadal tissue, GI EDG-3 Cardiovascular, Leukocyte EDG-4 Leukocyte,Testes EDG-5 Cardiovascular, CNS, Gonadal tissue, Placenta EDG-6Lymphoid, Hematopoietic tissue EDG-7 Heart, Pancreas, Prostate, TestesEDG-8 Brain PSP24 CNS

PLGFs activate multiple G-protein-mediated signal transduction events.These processes are mediated through the heterotrimeric G-proteinfamilies G_(q/11), G_(i/0), and G_(12/13) (Moolenaar, 1997; Spiegel andMilstein, 1995; Gohla, et al., 1998).

The G_(q/11) pathway is responsible for phospholipase C (PLC)activation, which in turn induces inositol triphosphate (IP₃) productionwith subsequent mobilization of Ca²⁺ in a wide variety of cells(Tokumura, 1995). In some cells, this response is PTX-sensitive,implying that there is involvement of multiple PTX-sensitive andinsensitive pathways (Tigyi et al., 1996). This pathway is alsoresponsible for the diacyl glycerol (DAG)-mediated activation of proteinkinase C (PKC). PKC activates cellular phospholipase D (PLD), which isresponsible for the hydrolysis of phosphatidyl choline into free cholineand PA (van der Bend et al., 1992a). Also, PLC is capable of activatingMAP kinase directly, or via DAG activation of PKC in some cell types(Ghosh et al., 1997).

The mitogenic-signaling pathway is mediated through the G-proteinheterotrimeric G_(i/0) subunit. Transfection studies indicate that theG_(iβγ) dimer rather than the αi subunit is responsible for Ras-MAPkinase activation. The activation of Ras is preceded by thetransactivation of the receptor tyrosine kinases (RTKs) such as EGF(Cunnick et al., 1998) or PDGF receptors (Herrlich et al., 1998). Thetransactivated RTKS activate Ras, which leads to the activation of MAPkinases (ERK 1, 2) via Raf. The G_(iα) subunit, which is PTX-sensitive,inhibits adenylyl cyclase (AC), resulting in βγ dimer docking to aG-protein-coupled receptor kinase (GRKs) that phosphorylates anddesensitizes the receptor. The phosphorylated receptor is recruited byβ-arrestin, thus recruiting src kinase, which phosphorylates theEGF-receptor, generating its active conformation (Lin et al., 1997; Ahnet al., 1999; Luttrell et al., 1999). The transactivated RTKs, in turn,activate Ras, which leads to the activation of MAP kinases (ERK 1, 2)via Raf. The G_(iα) subunit, which is PTX-sensitive, inhibits AC,resulting in decreased levels of cyclic-AMP (cAMP). The oppositecellular effects by LPA, that is, mitogenesis and antimitogenesis, areaccompanied by opposing effects on the cAMP second messenger system.Mitogenesis is mediated through the G_(iα) pathway, which results indecreased levels of cAMP (van Corven et al., 1989; van Corven et al.,1992), whereas anti-mitogenesis is accompanied by a non-PTX sensitiveCa²⁺-dependent elevation of cAMP (Tigyi et al., 1994; Fischer et al.,1998).

In contrast, very little is known about the PTX-insensitive G_(12/13)signaling pathway, which leads to the rearrangement of theactin-cytoskeleton. This pathway may also involve the transactivation ofRTKs (Lin et al., 1997; Ahn et al., 1999; Luttrell et al., 1999; Gohlaet al., 1998) and converge on a small GTPase, Rho (Moolenaar, 1997).Much more is known about the down-stream signaling of Rho becausevarious protein partners have been isolated and identified. Rhoactivates Ser/Thr kinases, which phosphorylate, and as a result inhibit,myosin light chain phosphatase (MLC-phosphatase) (Kimura et al., 1996).This path results in the accumulation of the phosphorylated form of MLC,leading to cytoskeletal responses that lead to cellular effects likeretraction of neurites (Tigyi and Miledi, 1992; Tigyi et al., 1996; Dyeret al., 1992; Postma et al., 1996; Sato et al., 1997), induction ofstress fibers (Ridley and Hall, 1992; Gonda et al., 1999), stimulationof chemotaxis (Jalink et al., 1993a), cell migration (Zhou et al., 1995;Kimura et al., 1992), and tumor cell invasiveness (Imamura et al., 1993;Imamura et al., 1996). The PLGF-induced, Rho-mediated, tumor cellinvasiveness is blocked by C. botulinium C3-toxin, which specificallyribosylates Rho in an ADP-dependent mechanism (Imamura et al., 1996).

Rho also has the ability to stimulate DNA synthesis in quiescentfibroblasts (Machesky and Hall, 1996; Ridley, 1996). The expression ofRho family GTPase activates serum-response factor (SRF), which mediatesearly gene transcription (Hill et al., 1995). Furthermore, PLGF (LPA)induces tumor cell invasion (Imamura et al., 1996); however, it is stillunclear whether it involves cytoskeletal changes or gene transcription,or both.

By virtue of LPA/LPA receptor involvement in a number of cellularpathways and cell activities such as proliferation and/or migration, aswell as their implication in wound healing and cancer, it would bedesirable to identify novel compounds which are capable of acting,preferably selectively, as either antagonists or agonists at the LPAreceptors identified above.

There are currently very few synthetic or endogenous LPA receptorinhibitors which are known. Of the antagonists reported to date, themost work was done on SPH, SPP, N-palmitoyl-1-serine (Bittman et al.,1996), and N-palmitoyl-1-tyrosine (Bittman et al., 1996). It is knownthat the above-mentioned compounds inhibit LPA-induced chloride currentsin the Xenopus oocyte (Bittman et al., 1996; Zsiros et al., 1998).However, these compounds have not been studied in all cell systems. Itis also known that SPP inhibits tumor cell invasiveness, but it isuncertain whether SPP does so by being an inhibitor of LPA or via theactions of its own receptors. N-palmitoyl-1-serine andN-palmitoyl-1-tyrosine also inhibited LPA-induced platelet aggregation(Sugiura et al., 1994), but it remains to be seen whether thesecompounds act at the LPA receptor. Lysophosphatidyl glycerol (LPG) wasthe first lipid to show some degree of inhibition of LPA actions (vander Bend et al., 1992b), but it was not detectable in severalLPA-responsive cells types (Liliom et al., 1996). None of theseinhibitors was shown to selectively act at specific LPA receptors.

A polysulfonated compound, Suramin, was shown to inhibit LPA-induced DNAsynthesis in a reversible and dose-dependent manner. However, it wasshown that Suramin does not have any specificity towards the LPAreceptor and blocked the actions of LPA only at very high millimolar(mM) concentrations (van Corven et al., 1992).

The present invention is directed to addressing the lack of agents whichact as LPA agonists and LPA antagonists.

SUMMARY OF THE INVENTION

The present invention relates to compounds according to formula (I)

wherein,

at least one of X¹, X², and X³ is (HO)₂PS-Z¹-, or (HO)₂PO-Z²-P(OH)S-Z¹-,X¹ and X² are linked together as —O—PS(OH)—O—, or X¹ and X³ are linkedtogether as —O—PS(OH)—NH—;

at least one of X¹, X², and X³ is R¹—Y¹-A- with each being the same ordifferent when two of X¹, X², and X³ are R¹—Y¹-A-, or X² and X³ arelinked together as —N(H)—C(O)—N(R¹)—;

optionally, one of X¹, X², and X³ is H;

A is either a direct link, (CH₂)_(k) with k being an integer from 0 to30, or O;

Y¹ is —(CH₂)_(l)— with l being an integer from 1 to 30, —O—, —S—,

or —NR²—;

Z¹, is —(CH₂)_(m)—, —CF₂—, —CF₂(CH₂)_(m)—, or —O(CH₂)_(m)— with m beingan integer from 1 to 50, —C(R³)H—, —NH—, —O—, or —S—;

Z² is —(CH₂)_(n)— or —O(CH₂)_(n)— with n being an integer from 1 to 50or —O—;

Q¹ and Q² are independently H₂, ═NR⁴, ═O, or a combination of H and—NR⁵R⁶;

R¹, for each of X¹, X², or X³, is independently hydrogen, a straight orbranched-chain C1 to C30 alkyl, a straight or branched-chain C2 to C30alkenyl, an aromatic or heteroaromatic ring with or without mono-, di-,or tri-substitutions of the ring, an acyl including a C1 to C30 alkyl oran aromatic or heteroaromatic ring, an arylalkyl including straight orbranched-chain C1 to C30 alkyl, an aryloxyalkyl including straight orbranched-chain C1 to C30 alkyl,

R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are independently hydrogen, a straight orbranched-chain C1 to C30 alkyl, a straight or branched-chain C2 to C30alkenyl, an aromatic or heteroaromatic ring with or without mono-, di-,or tri-substitutions of the ring, an acyl including a C1 to C30 alkyl oraromatic or heteroaromatic ring, an arylalkyl including straight orbranched-chain C1 to C30 alky, or an aryloxyalkyl including straight orbranched-chain C1 to C30 alkyl.

Also disclosed are pharmaceutical compositions which include apharmaceutically-acceptable carrier and a compound of the presentinvention.

A further aspect of the present invention relates to a method ofinhibiting LPA activity on an LPA receptor which includes providing acompound of the present invention which has activity as an LPA receptorantagonist and contacting an LPA receptor with the compound underconditions effective to inhibit LPA-induced activity of the LPAreceptor.

Another aspect of the present invention relates to a method ofmodulating LPA receptor activity which includes providing a compound ofthe present invention which has activity as either an LPA receptoragonist or an LPA receptor antagonist and contacting an LPA receptorwith the compound under conditions effective to modulate the activity ofthe LPA receptor.

Still another aspect of the present invention relates to a method oftreating cancer which includes providing a compound of the presentinvention and administering an effective amount of the compound to apatient in a manner effective to treat cancer.

Yet another aspect of the present invention relates to a method ofenhancing cell proliferation which includes providing a compound thepresent invention which has activity as an agonist of an LPA receptorand contacting the LPA receptor on a cell with the compound in a mannereffective to enhance LPA receptor-induced proliferation of the cell.

A further aspect of the present invention relates to a method oftreating a wound which includes providing a compound of the presentinvention which has activity as an agonist of an LPA receptor anddelivering an effective amount of the compound to a wound site, wherethe compound binds to LPA receptors on cells that promote healing of thewound, thereby stimulating LPA receptor agonist-induced cellproliferation to promote wound healing.

A still further aspect of the present invention relates to a method ofmaking the compounds of the present invention. One approach for makingthe compounds of the present invention includes:

reacting (Y²O)₂PO-Z¹¹-Z¹³ or (Y²O)₂PO-Z¹²-P(OH)—O-Z¹¹-Z¹³, where

Z¹¹ is —(CH₂)_(m)—, CF₂—, —CF₂(CH₂)_(m)—, or —O(CH₂)_(m)— with m beingan integer from 1 to 50, —C(R³)H—, —NH—, or —S—;

Z¹² is —(CH₂)_(n)— or —(CH₂)_(n)— with n being an integer from 1 to 50or —O—,

Z¹³ is H or a first leaving group or -Z¹¹-Z¹³ together form the firstleaving group; and

Y² is H or a protecting group, with an intermediate compound accordingto formula (IX) in the presence of sulfur

where,

at least one of X¹¹, X¹², and X¹³ is R¹¹—Y¹¹-A- with each being the sameor different when two of X¹¹, X¹², and X¹³ are R¹¹—Y¹¹-A-, or X¹² andX¹³ are linked together as —N(H)—C(O)—N(R¹¹)—;

at least one of X¹¹, X¹², and X¹³ is OH, NH₂, SH, or a second leavinggroup;

optionally, one of X¹¹, X¹², and X¹³ is H;

A is either a direct link, (CH₂)_(k) with k being an integer from 0 to30, or O;

Y¹¹ is —(CH₂)_(l)— with l being an integer from 1 to 30, —O—,

—S—, or —NR¹²—;

Q¹ and Q² are independently H₂, ═NR¹³, ═O, a combination of H and—NR¹⁴R¹⁵;

R¹¹, for each of X¹¹, X¹², or X¹³, is independently hydrogen, a straightor branched-chain C1 to C30 alkyl, a straight or branched-chain C2 toC30 alkenyl, an aromatic or heteroaromatic ring with or without

and mono-, di-, or tri-substitutions of the ring, an acyl including a C1to C30 alkyl or an aromatic or heteroaromatic ring, an arylalkylincluding straight or branched-chain C1 to C30 alkyl, an aryloxyalkylincluding straight or branched-chain C1 to C30 alkyl,

R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ are independently hydrogen, a straightor branched-chain C1 to C30 alkyl, a straight or branched-chain C2 toC30 alkenyl, an aromatic or heteroaromatic ring with or without mono-,di-, or tri-substitutions of the ring, an acyl including a C1 to C30alkyl or aromatic or heteroaromatic ring, an arylalkyl includingstraight or branched-chain C1 to C30 alkyl, or an aryloxyalkyl includingstraight or branched-chain C1 to C30 alkyl; followed by a de-protectionstep, if necessary, with both said reacting and the deprotection stepbeing performed under conditions effective to afford a compoundaccording to formula (I) where one or two of X¹, X², and X³ is(HO)₂PS-Z¹- or (HO)₂PS-Z²-P(OH)S-Z¹-.

Yet another aspect of the present invention relates to a method oftreating apoptosis or preserving or restoring function in a cell,tissue, or organ which includes: providing a compound of the presentinvention which has activity as an agonist of an LPA receptor; andcontacting a cell, tissue, or organ with an amount of the compound whichis effective to treat apoptosis or preserve or restore function in thecell, tissue, or organ.

A further aspect of the present invention relates to a method ofculturing cells which includes: culturing cells in a culture mediumwhich includes a compound of the present invention which has activity asan agonist of an LPA receptor and is present in an amount which iseffective to prevent apoptosis or preserve the cells in culture.

Another aspect of the present invention relates to a method ofpreserving an organ or tissue which includes: providing a compound ofthe present invention which has activity as an agonist of an LPAreceptor; and treating an organ or tissue with a solution comprising thecompound in an amount which is effective to preserve the organ or tissuefunction.

A related aspect of the present invention relates to an alternativemethod of preserving an organ or tissue which includes: providing acompound of the present invention which has activity as an agonist of anLPA receptor; and administering to a recipient of a transplanted organor tissue an amount of the compound which is effective to preserve theorgan or tissue function

A still further aspect of the present invention relates to a method oftreating a dermatological condition which includes: providing a compoundof the present invention which has activity as an LPA receptor agonist;and topically administering a composition comprising the compound to apatient, the compound being present in an amount which is effective totreat the dermatological condition

The compounds of the present invention which have been identified hereinas being either agonists or antagonists of one or more LPA receptorsfind uses to inhibit or enhance, respectively, biochemical pathwaysmediated by LPA receptor signaling. By modulating LPA receptorsignaling, the antagonists and agonists find specific and substantialuses as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates three reaction schemes used to prepare fatty acidphosphates (Scheme 1), fatty acid thiophosphonates (Scheme 2), anddifluorophosphonates (Scheme 3).

FIGS. 2A-D are graphs illustrating the effects of modified C-14 analogson RH7777 cells stably transfected with LPA₁₋₃ receptors. 200 nM of LPA18:1 was co-applied with increasing concentrations of C-14 analogs toRH7777 cells stably expressing LPA₁ and LPA₃. Increasing concentrationsof different C-14 analogs were applied to measure their agonisticproperties at LPA₂. Data points represent average of four measurements.FIG. 2A shows inhibition of the LPA response by C-14 analogs at LPA₁;FIG. 2B shows activation of LPA₂ by C-14 FAP analogs; FIG. 2C showsinhibition of the LPA response by C-14 phosphonate 9c at LPA₂; and FIG.2D shows inhibition of the LPA response by C-14 analogs at LPA₃.

FIGS. 3A-C are graphs illustrating that oleoyl-thiophosphate (8 g) is anagonist at LPA₁, LPA₂ and LPA₃ receptors expressed in RH7777 cells.Intracellular Ca²⁺ transients were measured in response to theapplication of increasing concentrations of 8 g and compared totransients elicited by the corresponding amount of LPA 18:1. Data pointsrepresent the average of four measurements. Dose-response relationshipsfor LPA 18:1 and 8 g in RH7777 cells expressing LPA₁ (FIG. 3A), LPA₂(FIG. 3B), and LPA₃ (FIG. 3C).

FIG. 4 is a bar graph depicting the results of in vitro PPARγ activationby selected compounds in CV1 cells transfected with PPARγ andPPRE-Acox-Rluc reporter gene. Comparing the effects with theRosiglitazone, a known PPARγ agonist, CV1 cells were treated with 1%DMSO or 10 μM of test compound dissolved in DMSO for 20 h. Luciferaseand β-galactosidase activities (mean ±SEM) were measured in the celllysate (n=4). *P<0.05 and **P<0.01, significant differences over vehiclecontrol.

FIG. 5 is a graph illustrating LPA and FAP18:1d9 thiophosphate (8 g)dose-dependently inhibit DNA fragmentation induced by Campothotecin (20μM).

FIG. 6 is a graph illustrating that FAP 18:1d9 thiophosphate (8 g)enhances crypt survival.

FIG. 7 is a graph illustrating the dose-dependent enhancement of cryptsurvival in FAP 18:1d9-treated mice.

FIG. 8 is a graph demonstrating that FAP 18:1d9 elicits dose-dependentcrypt survival in the ileum and jejunum of γ-irradiated mice.

FIG. 9 illustrates a synthesis scheme for preparing thiophosphoric acidesters containing an arylalkyl R¹ group when Y¹ is also an alkyl.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to a compound according toformula (I)

wherein,

at least one of X¹, X², and X³ is (HO)₂PS-Z¹-, or (HO)₂PS-Z²-P(OH)S-Z¹-,X¹ and X² are linked together as —O—PS(OH)—O—, or X¹ and X³ are linkedtogether as —O—PS(OH)—NH—;

at least one of X¹, X², and X³ is R¹—Y¹-A- with each being the same ordifferent when two of X¹, X², and X³ are R¹—Y¹-A-, or X² and X³ arelinked together as —N(H)—C(O)—N(R¹)—;

optionally, one of X¹, X², and X³ is H;

A is either a direct link, (CH₂)_(k) with k being an integer from 0 to30, or —O—;

Y¹ is —(CH₂)_(l)— with l being an integer from 1 to 30, —O—,

—S—, or —NR²—;

Z¹ is —(CH₂)_(m)—, —CF₂—, —CF₂(CH₂)_(m)—, or —O(CH₂)_(m)— with m beingan integer from 1 to 50, —C(R³)H—, —NH—, —O—, or —S—;

Z² is —(CH₂)_(n)— or —O(CH₂)_(n)— with n being an integer from 1 to 50or —O—;

Q¹ and Q² are independently H₂, ═NR⁴, ═O, a combination of H and —NR⁵R⁶;

R¹, for each of X¹, X², or X³, is independently hydrogen, a straight orbranched-chain C1 to C30 alkyl, a straight or branched-chain C2 to C30alkenyl, an aromatic or heteroaromatic ring with or without mono-, di-,or tri-substitutions of the ring, an acyl including a C1 to C30 alkyl oran aromatic or heteroaromatic ring, an arylalkyl including straight orbranched-chain C1 to C30 alkyl, an aryloxyalkyl including straight orbranched-chain C1 to C30 alkyl,

R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are independently hydrogen, a straight orbranched-chain C1 to C30 alkyl, a straight or branched-chain C2 to C30alkenyl, an aromatic or heteroaromatic ring with or without mono-, di-,or tri-substitutions of the ring, an acyl including a C1 to C30 alkyl oraromatic or heteroaromatic ring, an arylalkyl including straight orbranched-chain C1 to C30 alkyl, or an aryloxyalkyl including straight orbranched-chain C1 to C30 alkyl.

For each of the above-identified R groups (e.g., R¹-R⁸), straight chainalkyls have the formula —(CH₂)_(x)CH₃ where x is from 0 to 29; branchedchain alkyls have the formula as defined above for straight chain alkyl,except that one or more CH₂ groups are replaced by CHW groups where W isan alkyl side chain; straight chain alkenyls have the formula—(CH₂)_(Xa)CH═CH(CH₂)_(Xb)CH₃ where Xa and Xb each are from 0 to 27 and(Xa+Xb) is not more than 27; and branched chain alkenyls have theformula as defined above for straight chain alkenyl, except that one ormore CH₂ groups are replaced by CHW groups or a CH group is replaced bya CW group, where W is an alkyl side chain. Preferred hydrocarbon groupsare preferably between about 8 to about 18 carbon atoms in length, morepreferably between about 10 to about 16 carbon atoms in length, and maycontain one or more double bonds.

Aromatic or heteroaromatic rings include, without limitation, phenyls,indenes, pyrroles, imidazoles, oxazoles, pyrrazoles, pyridines,pyrimidines, pyrrolidines, piperidines, thiophenes, furans, napthals,bi-phenyls, and indoles. The aromatic or heteroaromatic rings caninclude mono-, di-, or tri-substitutions of the ring located at theortho, meta, or para positions on the rings relative to where the ringbinds to the Y¹ group of the R¹—Y¹-A- chain. Substitutions on the ringscan include, without limitation, alkyl, alkoxy, amine (includingsecondary or tertiary amines), alkylamine, amide, alkylamide, acids,alcohols.

Acyl groups can include either alkyl alkenyl, or aromatic orheteroaromatic rings as described above.

Arylalkyl and aryloxyalkyl groups can include, without limitation,straight or branched-chain C1 to C30 alkyl groups as described above,with the alkyl group binding to the Y¹ group of the R¹—Y¹-A- chain.

Exemplary compounds according to formula (I) are the subclass compoundsaccording to formulae (II)-(VII) below.

In the structures of formulae (II)A and (II)B, Q¹ and Q² are both H₂;one of X¹, X², and X³ is (HO)₂PS-Z²-P(OH)S-Z¹-, with Z¹ and Z² being O;and two of X¹, X², and X³ are R¹—Y¹-A-, with A being a direct link andY¹ being O for each. Each R¹ is defined independently as above forformula (I).

In the structures of formula (III), Q¹ is H₂; Q² is —O—: X¹ is(HO)₂PO-Z¹-, with Z¹ being O; and X² and X³ are R¹—Y¹-A-, with A being adirect link and Y¹ being —NH— for each. Each R¹ is defined independentlyas above for formula (I). Preferred species of within the scope offormula III are where X³ is —NH₂ and X² is —NHR¹ with R¹ being a C10 toC18 alkyl, more preferably either a C14 alkyl or a C18 alkyl; or whereX³ is —NHR¹ with R¹ being an acetyl group and X² is —NHR¹ with R¹ beinga C14 alkyl.

In the structures of formula (IV), Q¹ is ═NR⁴; Q² is H₂; X¹ and X² arelinked together as —O—PO(OH)—O—; and X³ is R¹—Y¹-A-, with A being adirect link and Y¹ being —NH—. R¹ and R⁴ are as defined above forformula (I).

In the structures of formulae (V)A and (V)B, Q¹ and Q² are both H₂; twoof X¹, X², and X³ are (HO)₂PO-Z¹-, with Z¹ being O for each; and one ofX¹, X², and X³ is R¹—Y¹-A-, with A being a direct link and Y¹ being —O—.R¹ is as defined above for formula (I). Preferred species within thescope of formulae (V)A and (V)B include the compounds where R¹ is anacyl including a C21 alkyl or where R¹ is a C18 alkyl.

The compounds according to formula (I), as well as the subgenuscompounds according to formulae (II)A, (II)B, (III), (IV), (V)A, and(V)B, may be prepared using the synthesis schemes described inPCT/US01/08729, filed Mar. 19, 2001, (incorporated by reference in itsentirety) except that phosphoramidate or pyrophosphates can be reactedin the presence of sulfur (with reflux) to obtain the thio-substitutedderivatives.

In the compounds according to formula (VI), Q¹ and Q² are both H₂; oneof X¹ and X² is (HO)₂PS-Z¹-, with Z¹ being O; and one of X¹, X², and X³is R¹—Y¹-A-, with A being a direct link and Y¹ being —CH₂—. R¹ is asdefined above for formula (I). Preferred R¹ groups are saturated andunsaturated C2 to C24 hydrocarbons, both straight and branched chain,and arylakyl groups containing C2 to C24 hydrocarbons; most preferred R¹groups are saturated and unsaturated C4 to C18 hydrocarbons. A preferredcompound according to formula VI is thiophosphoric acid O-octadec-9-enylester (8 g; also referred to as FAP 18:1d9).

The synthesis of thiophosphonates according to formula (VI) is outlinedin scheme 2 of FIG. 1. The protected thiophosphoric acidO,O′-bis-(2-cyano-ethyl) ester O″-alkyl/alkenyl esters can besynthesized using a modified method of Haines et al. (1996).Commercially available fatty alcohols (6a-g) can be treated with amixture of 1H-tetrazole and bis(2-cyanoethyl)-N,Ndiisopropylphosphoramidite in anhydrous methylene chloride followed by reflux inthe presence of elemental sulfur to give bis-cyanoethyl protected fattyalcohol thiophosphates (7a-g). These protected thiophosphates can betreated with methanolic KOH, followed by acidification to yield therequired thiophosphates (8a-g).

In the structures of formulae (VII)A and (VII)B, Q¹ and Q² are both H2;one of X¹, X², and X³ is (HO)₂PS-Z¹- with Z¹ being O; and two of X¹, X²,and X³ are R¹—Y¹-A-, with A being a direct link and Y¹ being O for each.Each R¹ is defined independently as above for formula (I).

Preferred R¹ groups are saturated and unsaturated C6 to C24hydrocarbons, both straight and branched chain; most preferred R¹ groupsare saturated and unsaturated C8 to C18 hydrocarbons. Two preferredcompounds according to group (VII)A are the (R) and (S) enantiomerswhere both R¹ groups are saturated octyl groups. The (R) enantiomer is apartial LPA₁ agonist (EC₅₀: 695 nM), a transient partial LPA₂ agonist(EC₅₀: 1.02 μM), and a full LPA₃ (EC₅₀: 3 nM) agonist. The (S)enantiomer is an agonist of the LPA₁ and LPA₃ receptors (IC₅₀ 328 nM forLPA₁ and IC₅₀ 184 nM for LPA, (both for 200 nM LPA)).

The compounds of formulae (VII)A and (VII)B can be prepared using thesynthesis schemes described in PCT/US01/08729, filed Mar. 19, 2001,which is hereby incorporated by reference in its entirety, except thatphosphoramidate can be reacted in the presence of sulfur (with reflux)to obtain the thio-substituted derivatives.

In the compounds according to formula (VIII), Q¹ and Q² are both H₂; oneof X¹ and X² is (HO)₂PS-Z¹-, with Z¹ being CF₂; and one of X¹, X², andX³ is R¹—Y¹-A-, with A being a direct link and Y¹ being —CH₂—. R¹ is asdefined above for formula (I).

Preferred R¹ groups are saturated and unsaturated C2 to C20hydrocarbons, both straight and branched chain; most preferred R¹ groupsare saturated and unsaturated C4 to C12 hydrocarbons.

The synthesis of difluorothiophosphonates according to formula (VIII) isoutlined in Scheme 3 of FIG. 1. The tetradecyl difluorophosphonateanalog was synthesized in two steps (Scheme 3) using diethyldifluoromethanephosphonate as the starting material (Halazy et al.,1991). Diethyl difluoromethanephosphonate was treated with LDA at −78°C. followed by reacting the anion with tetradecyl bromide to give theprotected phosphonate 10. Compound 10 was deprotected usingbromotrimethyl silane to yield the required difluorophosphonate compound(11).

Thus, the non-cyclic compounds of the present invention can be preparedby reacting (Y²O)₂PO-Z¹¹-Z¹³, (Y²O)₂PO-Z¹²-P(OH)S-Z¹¹-Z¹³, where Z¹¹ is—(CH₂)_(m), —CF₂—, —CF₂(CH₂)_(m), or —O(CH₂)_(m)— with m being aninteger from 1 to 50, —C(R³)H—, or —O—, Z¹² is —(CH₂)_(n)— or—O(CH₂)_(n)— with n being an integer from 1 to 50 or —O—, Z¹³ is H or afirst leaving group or -Z¹¹-Z¹³ together to form the first leavinggroup, and Y² is H or a protecting group; with an intermediate compoundaccording to formula (IX) in the presence of sulfur, followed by ade-protection step, if necessary, both performed under conditionseffective to afford a compound according to formula (I) where one or twoof X¹, X², and X³ is (HO)₂PS-Z¹- or (HO)₂PS-Z²-P(OH)S-Z¹- with Z¹ and Z²being defined as above.

The intermediate compound of formula (IX) has the following structure:

wherein,

at least one of X¹¹, X¹², and X¹³ is R¹¹—Y¹¹-A- with each being the sameor different when two of X¹¹, X¹², and X¹³ are R¹¹—Y¹¹-A-, or X¹² andX¹³ are linked together as —N(H)—C(O)—N(R¹¹)—;

at least one of X¹¹, X¹², and X¹³ is OH, NH₂, SH, or a second leavinggroup;

optionally, one of X¹¹, X¹², and X¹³ is H;

A is either a direct link, (CH₂)_(k) with k being an integer from 0 to30, or O;

Y¹¹ is —(CH₂)_(l)— with l being an integer from 1 to 30, —O—,

Q¹ and Q² are independently H₂, ═NR¹³, ═O, a combination of H and—NR¹⁴R¹⁵;

R¹¹, for each of X¹¹, X¹², or X¹³, is independently hydrogen, a straightor branched-chain C1 to C30 alkyl, a straight or branched-chain C2 toC30 alkenyl, an aromatic or heteroaromatic ring with or without mono-,di-, or tri-substitutions of the ring, an acyl including a C1 to C30alkyl or an aromatic or heteroaromatic ring, an arylalkyl includingstraight or branched-chain C1 to C30 alkyl, an aryloxyalkyl includingstraight or branched-chain C1 to C30 alkyl,

R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ are independently hydrogen, a straightor branched-chain C1 to C30 alkyl, a straight or branched-chain C2 toC30 alkenyl, an aromatic or heteroaromatic ring with or without mono-,di-, or tri-substitutions of the ring, an acyl including a C1 to C30alkyl or aromatic or heteroaromatic ring, an arylalkyl includingstraight or branched-chain C1 to C30 alkyl, or an aryloxyalkyl includingstraight or branched-chain C1 to C30 alkyl.

Having prepared the LPA receptor agonists and antagonists of the presentinvention, such compounds can be used to prepare pharmaceuticalcompositions suitable for treatment of patients as describedhereinafter. Therefore, a further aspect of the present inventionrelates to a pharmaceutical composition that includes apharmaceutically-acceptable carrier and a compound of the presentinvention. The pharmaceutical composition can also include suitableexcipients, or stabilizers, and can be in solid or liquid form such as,tablets, capsules, powders, solutions, suspensions, or emulsions.Typically, the composition will contain from about 0.01 to 99 percent,preferably from about 20 to 75 percent of active compound(s), togetherwith the carrier, excipient, stabilizer, etc.

The solid unit dosage forms can be of the conventional type. The solidform can be a capsule, such as an ordinary gelatin type containing thecompounds of the present invention and a carrier, for example,lubricants and inert fillers such as, lactose, sucrose, or cornstarch.In another embodiment, these compounds are tableted with conventionaltablet bases such as lactose, sucrose, or cornstarch in combination withbinders like acacia, cornstarch, or gelatin, disintegrating agents, suchas cornstarch, potato starch, or alginic acid, and a lubricant, likestearic acid or magnesium stearate.

The compounds of the present invention may also be administered ininjectable or topically-applied dosages by solution or suspension ofthese materials in a physiologically acceptable diluent with apharmaceutical carrier. Such carriers include sterile liquids, such aswater and oils, with or without the addition of a surfactant and otherpharmaceutically and physiologically acceptable carrier, includingadjuvants, excipients or stabilizers. Illustrative oils are those ofpetroleum, animal, vegetable, or synthetic origin, for example, peanutoil, soybean oil, or mineral oil. In general, water, saline, aqueousdextrose and related sugar solution, and glycols, such as propyleneglycol or polyethylene glycol, are preferred liquid carriers,particularly for injectable solutions.

For use as aerosols, the compounds of the present invention in solutionor suspension may be packaged in a pressurized aerosol containertogether with suitable propellants, for example, hydrocarbon propellantslike propane, butane, or isobutane with conventional adjuvants. Thematerials of the present invention also may be administered in anon-pressurized form such as in a nebulizer or atomizer.

Depending upon the treatment being effected, the compounds of thepresent invention can be administered orally, topically, transdermally,parenterally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, intraocularly, intraarterially,intralesionally, or by application to mucous membranes, such as, that ofthe nose, throat, and bronchial tubes.

Compositions within the scope of this invention include all compositionswherein the compound of the present invention is contained in an amounteffective to achieve its intended purpose. While individual needs vary,determination of optimal ranges of effective amounts of each componentis within the skill of the art. Typical dosages comprise about 0.01 toabout 100 mg/kg body wt. The preferred dosages comprise about 0.1 toabout 100 mg/kg body wt. The most preferred dosages comprise about 1 toabout 100 mg/kg body wt. Treatment regimen for the administration of thecompounds of the present invention can also be determined readily bythose with ordinary skill in art.

Certain compounds of the present invention have been found to be usefulas agonists of LPA receptors while other compounds of the presentinvention have been found useful as antagonists of LPA receptors. Due totheir differences in activity, the various compounds find differentuses. The preferred animal subject of the present invention is a mammal.The invention is particularly useful in the treatment of human subjects.

One aspect of the present invention relates to a method of modulatingLPA receptor activity which includes providing a compound of the presentinvention which has activity as either an LPA receptor agonist or an LPAreceptor antagonist and contacting an LPA receptor with the compoundunder conditions effective to modulate the activity of the LPA receptor.

The LPA receptor is present on a cell which either normally expressesthe LPA receptor or has otherwise been transformed to express aparticular LPA receptor. Suitable LPA receptors include, withoutlimitation, EDG-2 (LPA₁), EDG-4 (LPA₂), EDG-7 (LPA₃), GPR23 (LPA₄)(Noguchi et al. 2003), and PSP-24 receptors. The tissues which containcells that normally express these receptors are indicated in Table 1.When contacting a cell with the LPA receptor agonist or LPA receptorantagonist of the present invention, the contacting can be carried outwhile the cell resides in vitro or in vivo.

To heterologously express these receptors in host cells which do notnormally express them, a nucleic acid molecule encoding one or more ofsuch receptors can be inserted in sense orientation into an expressionvector which includes appropriate transcription and translationsregulatory regions (i.e., promoter and transcription terminationsignals) and then host cells can be transformed with the expressionvector. The expression vector may integrate in the cellular genome orsimply be present as extra-chromosomal nuclear material. Expression canbe either constitutive or inducible, although constitutive expression issuitable for most purposes.

The nucleotide and amino acid sequences for EDG-2 is known and reportedin An et al. (1997b) and Genbank Accession No. U80811, which is herebyincorporated by reference. An EDG-2 (LPA₁) encoding nucleic acidmolecule has a nucleotide sequence according to SEQ. ID. No. 1 asfollows: atggctgcca tctctacttc catccctgta atttcacagc cccagttcacagccatgaat   60 gaaccacagt gcttctacca cgagtccatt gccttctttt ataaccgaagtggaaagcat  120 cttgccacag aatggaacac agtcagcaag ctggtgatgg gacttggaatcactgtttgt  180 atcttcatca tgttggccaa cctattggtc atggtggcaa tctatgtcaaccgccgcttc  240 cattttccta tttattacct aatggctaat ctggctgctg cagacttctttgctgggttg  300 gcctacttct atctcatgtt caacacagga cccaatactc ggagactgactgttagcaca  360 tggctcctgc gtcagggcct cattgacacc agcctgacgg catctgtggccaacttactg  420 gctattgcaa tcgagaggaa cattacggtt ttccgcatgc agctccacacacggatgagc  480 aaccggcggg tagtggtggt cattgtggtc acctggacta tggccatcgttatgggtgct  540 atacccagtg tgggctggaa ctgtatccgt gatattgaaa attgttccaacacggcaccc  600 ctctacagtg actctcactt agtcttctgg gccactttca acttggtgacctccgtggta  660 atggtggttc tctatgctca catctttggc tatgctcgcc agaggactatgagaatgtct  720 cggcatagtt ctggaccccg gcggaatcgg gataccatga tgagtcttctgaagactgtg  700 gccattgtgc ttggggcctt tatcatctgc tggacccctg gattggctttgtcacttcta  840 gacgtgtgct gtccacagtg cgacgtgctg gcctatgaga aattcttccttctccttgct  900 gaactcaact ctgccatgaa ccccatcatt tactcccacc gcgacaaagaaatgagcgcc  960 accctcaggc agatcctctg ctgccagcgc agtgagaacc ccaccggccccacagaaagc 1020 tcagaccgct cggcttcctc cctcaaccac accatcttgg ctggagttcacagcaatgac 1080 cactctgtgg tctag 1093

The encoded EDG-2 (LPA₁) receptor has an amino acid sequence accordingto SEQ. ID. No. 2 as follows: MAAISTSIPV ISQPQFTAMN EPQCFYNESIAFFYNRSGKH LAIEWNTVSK LVMGLGITVC  60 IFIMLANLLV MVAIYVNRRF HFPIYYLMANLAAADFFAGL AYFYLMFNTG PNTRRLTVST 120 WLLRQGLIDT SLTASVANLL AIAIERHITVFRMQLHTRMS NRRVVVVIVV IWTMAIVMGA 180 IPSVGWNCIC DIENCSNMAP LYSDSYLVFWAIFNLVTFVV MVVLYAHIFG YVRQRTMRMS 240 RHSSGPRRNR DTMMSLLKTV VIVLGAFIICWTPGLVLLLL DVCCPQCDVL AYEKFFLLLA 300 EFNSAMNPII YSYRDKEMSA TFRQILCCQRSENPTGPTES SDRSASSLNH TILAGVHSND 360 HSVV 364

The nucleotide and amino acid sequences for EDG-4 (LPA₂) is known andreported in An et al. (1998b) and Genbank Accession No. NM₀₀₄₇₂₀. AnEDG-4 encoding nucleic acid molecule has a nucleotide sequence accordingto SEQ. ID. No. 3 as follows: atggtcatca tgggccagtg ctactacaacgagaccatcg gcttcttcta taacaacagt   60 ggcaaagagc tcagctccca ctggcggcccaaggatgtgg tcgtggtggc accggggctg  120 accgccagcg tgctggcgct gctgaccaatctgctggtca tagcagccat cgcctccaac  180 cgccgcttcc accagcccat ctactacctgctcggcaatc tggccgcggc tgacctcttc  240 gcgggcgtgg cctacctctt cctcatgttccacactggtc cccgcacagc ccgactctca  300 cttgagggct ggttcctgcg gcagggcctgctggacacaa gcctcactgc gccggtggcc  360 acactgctgg ccatcgccgt ggagcggcaccgcagtgtga tggccgtgca gctgcacagc  420 cgcctgcccc gtggccgcgt ggtcatgctcattgtgggcg cgtgggtggc tgccctgggc  480 ctggggctgc tgcctgccCa ctcctggcactgcctctgtg ccctggaccg ctgctcacgc  540 atggcacccc tgctcagccg ctcctatttggccgtctggg ctctgtcgag cctgcttgtc  600 ttcatgctca tggtggctgt gtacacccgcattttcttct acgtgcggcg gcgagtgcag  660 cgcatggcag agcatgtcag ctgccacccccgctaccgag agaccacgct cagcctggtc  720 aagactgttg tcatcatcct gggggcgttcgtggtctgct ggacaccagg ccaggtggta  780 ctgctcctgg atggtttagg ctgtgagtcctgcaatgtcc tggctgtaga aaagtacttc  840 ctactgttgg ccgaggccaa ctcactggtcaatgctgctg tgtactcttg ccgagatgct  900 gagacgcgcc gcaccttccg ccgccttctctgctgcgcgt gcctccgcca gtccacccgc  960 gagtctgtcc actatacatc ctctgcccagggaggtgcca gcactcgcat cacgcttccc 1020 gagaacggcc acccactgat ggactccaccctttag 1056

The encoded EDG-4 (LPA₂) receptor has an amino acid sequence accordingto SEQ. ID. No. 4 as follows: MVIMGQCYYN ETIGFFYNNS GKELSSHWRPKDVVVVALGL TVSVLVLLTN LLVIAAIASN  60 RRFHQPIYYL LGNLAAADLF AGVAYLFLMFHTGPRTARLS LEGWFLRQGL LDTSLTASVA 120 TLLAIAVERH RSVMAVQLHS RLPRGRVVMLIVGVWVAALG LGLLPAHSWH CLCALDRCSR 180 MAPLLSRSYL AVWALSSLLV FLLMVAVYTRIFFYVRRRVQ RMAEHVSCHP RYRETTLSLV 240 KTVVIILGAF VVCWTPGQVV LLLDGLGCESCNVLAVEKYF LLLAEANSLV NAAVYSCRDA 300 EMRRTFRRLL CCACLRQSTR ESVHYTSSAQGGASTRIMLP ENGHPLMDST l 351

The nucleotide and amino acid sequences for EDG-7 (LPA₃) is known andreported in Bandoh et al. (1999) and Genbank Accession No. NM₀₁₂₁₅₂. AnEDG-7 encoding nucleic acid molecule has a nucleotide sequence accordingto SEQ. ID. No. 5 as follows: atgaatgagt gtcactatga caagcacatggacttttttt ataataggag caacactgat   60 actgtcgatg actggacagg aacaaagcttgtgattgttt tgtgtgttgg gacgtttttc  120 tgcctgttta tttttttttc taattctctggtcatcgcgg cagtgatcaa aaacagaaaa  180 tttcatttcc ccttctacta cctgttggctaatttagctg ctgccgattt cttcgctgga  240 attgcctatg tattcctgat gtttaacacaggcccagttt caaaaacttt gactgtcaac  300 cgctggtttc tccgtcaggg gcttctggacagtagcttga ctgcttccct caccaacttg  360 ctggttatcg ccgtggagag gcacatgtcaatcatgagga tgcgggtcca tagcaacctg  420 accaaaaaga gggtgacact gctcattttgcttgtctggg ccatcgccat ttttatgggg  480 gcggtcccca cactgggctg gaattgcctctgcaacatct ctgcctgctc ttccctggcc  540 cccatttaca gcaggagtta ccttgttttctggacagtgt ccaacctcat ggccttcctc  600 acatgctttg tggtgtacct gcggatctacgtgtacgtca agaggaaaac caacgtcttg  660 tctccgcata caagtgggtc catcagccgccggaggacac ccatgaagct aatgaagacg  720 gtgatgactg tcttaggggc gtttgtggtatgctggaccc cgggcctggt ggttctgctc  780 ctcgacggcc tgaactgcag gcagtgtggcgtgcagcatg tgaaaaggtg gttcctgctg  840 ctggcgctgc tcaactccgt cgtgaaccccatcatctact cctacaagga cgaggacatg  900 tatggcacca tgaagaagat gatctgctgattctctcagg agaacccaga gaggcgtccc  960 tctcgcatcc cctccacagt cctcagcaggagtgacacag gcagacagta catagaggat 1020 agtattagcc aaggtgcagt ctgcaataaaagcacttcct aa 1062

The encoded EDG-7 (LPA₃) receptor has an amino acid sequence accordingto SEQ. ID. No. 6 as follows: MNECHYDKHM DFFYNRSNTD TVDDWTGTKLVIVLCVGTFF CLFIFFSNSL VIAAVIKNRK  60 FHFPFYYLLA NLAAADFFAG IAYVFLMFNTGPVSKTLTVN RWFLRQGLLD SSLTASLTNL 120 LVIAVERHMS IMRMRVHSNL TKKRVTLLILLVWAIAIFMG AVPTLGWNCL CNISACSSLA 180 PIYSRSYLVF WTVSNLMAFL IMVVVYLRIYVYVKRKTNVL SPHTSGSISR RRTPMKLMKT 240 VMTVLGAFVV CWTPGLVVLL LDGLNCRQCGVQHVKRWFLL LALLNSVVNP IIYSYKDEDM 300 YGTMKKMICC FSQENPERRP SRIPSTVLSRSDTGSQYIED SISQGACCNK STS 353

The nucleotide and amino acid sequences for PSP-24 is known and reportedin Kawasawa et al. (2000) and Genbank Accession No. AB030566. A PSP-24encoding nucleic acid molecule has a nucleotide sequence according toSEQ. ID. No. 7 as follows: atggtcttct cggcagtgtt gactgcgttc cataccgggacatccaacac aacatttgtc   60 gtgtatgaaa acacctacat gaatattaca ctccctccaccattccagca tcctgacctc  120 agtccattgc ttagatatag ttttgaaacc atggctcccactggtttgag ttccttgacc  180 gtgaatagta cagctgtgcc cacaacacca gcagcatttaagagcctaaa cttgcctctt  240 cagatcaccc tttctgctat aatgatattc attctgtttgtgtcttttct tgggaacttg  300 gttgtttgcc tcatggttta ccaaaaagct gccatgaggtctgcaattaa catcctcctt  360 gccagcctag cttttgcaga catgttgctt gcagtgctgaacatgccctt tgccctggta  420 actattctta ctacccgatg gatttttggg aaattcttctgtagggtatc tgctatgttt  480 ttctggttat ttgtgataga aggagtagcc atcctgctcatcattagcat agataggttc  540 cttattatag tccagaggca ggataagcta aacccatatagagctaaggt tctgattgca  600 gtttcttggg caacttcctt ttgtgtagct tttcctttagccgtaggaaa ccccgacctg  660 cagatacctt cccgagctcc ccagtgtgtg tttgggtacacaaccaatcc aggataccag  720 gcttatgcga ttttgatttc tctcatttct ttcttcatacccttcctggt aatactgtac  780 tcatttatgg gcacacccaa cacccttcgg cacaatgccttgaggaccca tagctaccct  840 gaaggtatat gcctcagcca ggccagcaaa ctgggtcccatgagtctgca gagacctttc  900 cagatgagca ccgacatggg ccttaaaaca cgtgcctccaccactatttt gaccctcttt  960 gctgtcttca ttgtctgctg ggccccattc accacccacagccccgtggc aacattcagt 1020 aagcactttc actatcagca caactttttc gagattagcacccggctact gtggctccgc 1080 taccccaagt ctgcattgaa tccgctgatc tactactggaggattaagaa attccatgat 1140 gcctgcctgg acacgatgcc taagtccctc aagtttttgccgcagctccc tggtcacaca 1200 aagcgacgga tacgtcccag tgctgcctat gtgtgtggggaacatcggac ggtggtgtga 1260

The encoded PSP-24 receptor has an amino acid sequence according to SEQ.ID. No. 8 as follows: MFFSAVLTAF HTGTSNTTFV VYENTYMNIT LPPPFQHPDLSPLLRYSFET MAPTGLSSLT  60 VNSTAVPTTP AAFKSLNLPL QITLSAIMIF ILFVSFLGNLVVCLMVYQKA AMRSAINILL 120 ASLAFADMLL AVLNMPFALV TILTTRWIFG KFFCRVSAMFFWLFVIEGVA ILLIISIDRF 180 LIIVQRQDKL NPYRAKVLIA VSWATSFCVA FPLAVGNPDLQIPSPAPQCV FGYTTMPGYQ 240 AYVILISLIS FFIPFLVILY SFMGILNTLR HNALRIHSYPEGICLSQASK LGLMSLQRPF 300 QMSIDMGFKT RAFTTILILF AVFIVCWAPF TTYSLVATFSKHFYYQHNFF EISTWLLWLC 360 YLKSALNPLI YYWRIKKFHD ACLDMMPKSF KFLPQLPGHTKRRIRPSAVY VCGEHRTVV 419

LPA receptor agonists will characteristically induce LPA-like activityfrom an LPA receptor, which can be measured either chemically, e.g.,Ca²+ or Cl⁻ current in oocytes, or by examining changes in cellmorphology, mobility, proliferation, etc. In contrast, LPA receptorantagonists will characteristically block LPA-like activity from an LPAreceptor. This too can be measured either chemically, e.g., Ca²+ or Cl⁻current in oocytes, or by examining changes in cell morphology,mobility, proliferation, etc.

By virtue of the compounds of the present invention acting as LPAreceptor antagonists, the present invention also relates to a method ofinhibiting LPA-induced activity on an LPA receptor. This method includesproviding a compound of the present invention which has activity as anLPA receptor antagonist and contacting an LPA receptor with the compoundunder conditions effective to inhibit LPA-induced activity of the LPAreceptor. The LPA receptor can be as defined above. The LPA receptor ispresent on a cell which normally expresses the receptor or whichheterologously expresses the receptor. The contacting of the LPAreceptor with the compound of the present invention can be performedeither in vitro or in vivo.

As noted above, LPA is a signaling molecule involved in a number ofdifferent cellular pathways which involve signaling through LPAreceptors, including those LPA receptors described above. Therefore, itis expected that the compounds of the present invention will modulatethe effects of LPA on cellular behavior, either by acting as LPAreceptor antagonists or LPA receptor agonists.

One aspect of the present invention relates to a method of treatingcancer which includes providing a compound of the present invention andadministering an effective amount of the compound to a patient in amanner effective to treat cancer. The types of cancer which can betreated with the compounds of the present invention include thosecancers characterized by cancer cells whose behavior is attributable atleast in part to LPA-mediated activity. Typically, these types of cancerare characterized by cancer cells which express one or more types of LPAreceptors. Exemplary forms of cancer include, without limitation,prostate cancer, ovarian cancer, and bladder cancer.

The compounds of the present invention which are particularly useful forcancer treatment are the LPA receptor antagonists.

When administering the compounds of the present invention, they can beadministered systemically or, alternatively, they can be administereddirectly to a specific site where cancer cells are present. Thus,administering can be accomplished in any manner effective for deliveringthe compound to cancer cells. LPA receptor antagonists, upon binding toLPA receptors, inhibit proliferation or metastasis of the cancer cellsor destroy those cancer cells. As shown in Example 12, several LPAantagonist compounds of the present invention were cytotoxic to prostatecancer cell lines which express one or more LPA receptors of the typedescribed above.

When the LPA antagonist compounds or pharmaceutical compositions of thepresent invention are administered to treat cancer, the pharmaceuticalcomposition can also contain, or can be administered in conjunctionwith, other therapeutic agents or treatment regimen presently known orhereafter developed for the treatment of various types of cancer.

Cancer invasion is a complex multistep process in which individual cellsor cell clusters detach from the primary tumor and reach the systemiccirculation or the lymphatics to spread to different organs (Liotta etal., 1987). During this process, tumor cells must arrest in capillaries,extravasate, and migrate into the stroma of the tissue to make secondaryfoci. First, tumor cells must recognize signals on the endothelial cellthat arrest them from the circulation. Second, tumor cells must attachto the basement membrane glycoprotein laminin via the cell surfacelaminin receptors. Following attachment to the basement membrane, tumorcells secrete proteases to degrade the basement membrane. Followingattachment and local proteolysis, the third step of invasion is tumorcell migration. Cell motility plays a central role in tumor cellinvasion and metastasis. The relationship between motility of tumorcells in vitro and the metastatic behavior in animal experimentsindicates a strong direct correlation (Hoffman-Wellenhof et al., 1995).It is a well-documented fact that PLGFs promote proliferation andincrease invasiveness of cancer cell in vitro. Imamura and colleaguesestablished that cancer cells require serum factors for their invasion(Imamura et al., 1991), and later identified LPA as the most importantserum component that is fully capable of restoring tumor cell invasionin serum-free systems (Xu et al., 1995a; Imamura et al., 1993; Mukai etal., 1993).

It has been shown that PLGFR are expressed in ovarian cancer cell lines;namely, OCC1 and HEY cells. Specifically, RT-PCR analyses show thepresence of EDG-2 and EDG-7 receptors in these cell lines. Recently, Imet al. (2000) demonstrated that EDG-7 is expressed in prostate cancercell lines; namely, PC-3 and LNCaP cells. RT-PCR analysis on theprostate cancer cell lines DU-145, PC-3, and LNCaP lines showed thatEDG-2, 4, 5, and EDG-7 are present in all three prostate cancer celllines, whereas EDG-3 is present in LNCaP and DU-145 prostate cancer celllines.

Another aspect of the present invention relates to a method of enhancingcell proliferation. This method of enhancing cell proliferation includesthe steps of providing a compound of the present invention which hasactivity as an agonist of an LPA receptor and contacting the LPAreceptor on a cell with the compound in a manner effective to enhanceLPA receptor-induced proliferation of the cell.

In addition to the roles that LPA plays in modulating cancer cellactivity, there is strong evidence to suggest that LPA also has aphysiological role in natural wound healing. At wound sites, LPA derivedfrom activated platelets is believed to be responsible, at least inpart, for stimulating cell proliferation at the site of injury andinflammation possibly in synchronization with other platelet-derivedfactors (Balazs et al., 2000). Moreover, LPA by itself stimulatesplatelet aggregation, which may in turn be the factor that initiates anelement of positive feedback to the initial aggregatory response(Schumacher et al., 1979; Tokumura et al., 1981; Gerrard et al., 1979;Simon et al., 1982).

Due to the role of LPA in cell proliferation, compounds having LPAreceptor agonist activity can be used in a manner effective to promotewound healing. Accordingly, another aspect of the present inventionrelates to a method of treating a wound. This method is carried out byproviding a compound of the present invention which has activity as anagonist of an LPA receptor and delivering an effective amount of thecompound to a wound site, where the compound binds to LPA receptors oncells that promote healing of the wound, thereby stimulating LPAreceptor agonist-induced cell proliferation to promote wound healing.

The primary goal in the treatment of wounds is to achieve wound closure.Open cutaneous wounds represent one major category of wounds and includeburn wounds, neuropathic ulcers, pressure sores, venous stasis ulcers,and diabetic ulcers. Open cutaneous wounds routinely heal by a processwhich comprises six major components: i) inflammation, ii) fibroblastproliferation, iii) blood vessel proliferation, iv) connective tissuesynthesis v) epithelialization, and vi) wound contraction. Wound healingis impaired when these components, either individually or as a whole, donot function properly. Numerous factors can affect wound healing,including malnutrition, infection, pharmacological agents (e.g.,actinomycin and steroids), diabetes, and advanced age (see Hunt andGoodson, 1988).

Phospholipids have been demonstrated to be important regulators of cellactivity, including mitogenesis (Xu et al., 1995b), apoptosis, celladhesion, and regulation of gene expression. Specifically, for example,LPA elicits growth factor-like effects on cell proliferation (Moolenaar,1996) and cell migration (Imamura et al., 1993). It has also beensuggested that LPA plays a role in wound healing and regeneration (Tigyiand Miledi, 1992).

In general, agents which promote a more rapid influx of fibroblasts,endothelial and epithelial cells into wounds should increase the rate atwhich wounds heal.

In vitro systems model different components of the wound healingprocess, for example the return of cells to a “wounded” confluentmonolayer of tissue culture cells, such as fibroblasts (Verrier et al.,1986), endothelial cells (Miyata et al., 1990) or epithelial cells(Kartha et al., 1992). Other systems permit the measurement ofendothelial cell migration and/or proliferation (Muller et al., 1987;Sato et al., 1988).

In vivo models for wound healing are also well-known in the art,including wounded pig epidermis (Ohkawara et al., 1977) or drug-inducedoral mucosal lesions in the hamster cheek pouch (Cherrick et al., 1974).

The compounds of the present invention which are effective in woundhealing can also be administered in combination, i.e., in thepharmaceutical composition of the present invention or simultaneouslyadministered via different routes, with a medicament selected from thegroup consisting of an antibacterial agent, an antiviral agent, anantifungal agent, an antiparasitic agent, an antiinflammatory agent, ananalgesic agent, an antipruritic agent, or a combination thereof.

For wound healing, a preferred mode of administration is by the topicalroute. However, alternatively, or concurrently, the agent may beadministered by parenteral, subcutaneous, intravenous, intramuscular,intraperitoneal or transdermal routes. Alternatively, or concurrently,administration may be by the oral route. The dosage administered will bedependent upon the age, health, and weight of the recipient, kind ofconcurrent treatment, if any, frequency of treatment, and the nature ofthe effect desired.

For the preferred topical applications, especially for treatment ofhumans and animals having a wound, it is preferred to administer aneffective amount of a compound according to the present invention to thewounded area, e.g., skin surfaces. This amount will generally range fromabout 0.001 mg to about 1 g per application, depending upon the area tobe treated, the severity of the symptoms, and the nature of the topicalvehicle employed. A preferred topical preparation is an ointment whereinabout 0.01 to about 50 mg of active ingredient is used per ml ofointment base, such as PEG-1000.

The present invention further provides methods of inhibiting apoptosisor preserving or restoring cell, tissue or organ function. This methodis carried out by providing a compound of the present invention whichhas activity as an agonist of an LPA receptor and contacting a cell,tissue, or organ with an amount of the compound which is effective toinhibit apoptosis, or preserve or restore function in the cell, tissue,or organ. The contacting can be carried out in vitro (i.e., during cellculture or organ or tissue transfer) or in vivo (i.e., by administeringthe effective amount of the compound to a patient as indicated below).

Various indications which can be treated, include, but are not limitedto, those related to apoptosis, ischemia, traumatic injury, andreperfusion damage. Those conditions related to apoptosis include, butare not limited to, dermatological effects of aging, the effects ofreperfusion after an ischemic event, immunosuppression, gastrointestinalperturbations, cardiovascular disorders, rejection of tissuetransplantation, wound healing, and Alzheimer's disease. The treatmentcan also diminish the apoptosis-related problems associated with viralinfection, chemotherapeutic agents, radiation, and immunosuppressivedrugs. These stimuli trigger apoptosis in a variety of disorders,including, but not limited to, those of the digestive tract tissues.

A preferred compound for practicing this aspect of the present inventionis compound 8g, particularly with respect to the protection ofgastroendothelial cells against chemotherapeutic- or radiation-inducedapoptosis as described in the Examples herein.

The treatments are also suitable during all phases of organtransplantation. The compounds having agonist activity on an LPAreceptor can be used to prepare the organ by administering an amount ofthe compound to the donor effective to stabilize or preserve the organ.The organ can be perfused and/or preserved in OPS containing thecompound. The organ recipient can then be administered an amount of thecompound effective to enhance organ stability and function. Thecompositions are also particularly suitable for use in treatingcardioplegia, whether related to transplantation or other surgicalintervention.

Gastrointestinal tissue damage includes, but is not limited to, damageto the lining of the gut, severe chronic ulcers, colitis, radiationinduced damage, chemotherapy induced damage, and the perturbation of thegastrointestinal tract caused by parasites, and diarrhea from any othercause. Various viral and bacterial infections are known to result ingastrointestinal perturbations. The compounds having agonist activity onan LPA receptor are also suitable for use in treatment of the sideeffects associated with these infections. Such compounds areparticularly suited for use in ameliorating the gastrointestinaldisturbances associated with chemotherapy. Thus, such compounds aresuitable for use not only in preventing the diarrhea associated withchemotherapy but also the nausea.

These compounds are particularly suited to treatment of variousgastrointestinal conditions in animals, including, but not limited tolivestock and domesticated animals. Such conditions, particularlydiarrhea, account for the loss of many calves and puppies to dehydrationand malnutrition. Treatment of gastrointestinal conditions is preferablyby gastrointestinal administration. In the case of cattle anddomesticated animals, an effective amount of these compounds can beconveniently mixed in with the feed. In humans, administration can be byany method known in the art of gastrointestinal administration.Preferably, administration is oral.

In addition, the compounds having agonist activity on an LPA receptorcan be administered to immunodeficient patients, particularlyHIV-positive patients, to prevent or at least mitigate apoptotic deathof T cells associated with the condition, which results in theexacerbation of immunodeficiencies as seen in patients with AIDS.Preferably, administration to such patients is parenteral, but can alsobe transdermal or gastrointestinal.

The compounds having agonist activity on an LPA receptor can also beadministered to treat apoptosis associated with reperfusion damageinvolved in a variety of conditions, including, but not limited to,coronary artery obstruction; cerebral infarction; spinal/head trauma andconcomitant severe paralysis; reperfusion damage due to other insultssuch as frostbite, coronary angioplasty, blood vessel attachment, limbattachment, organ attachment and kidney reperfusion.

Myocardial and cerebral infarctions (stroke) are caused generally by asudden insufficiency of arterial or venous blood supply due to emboli,thrombi, or pressure that produces a macroscopic area of necrosis; theheart, brain, spleen, kidney, intestine, lung and testes are likely tobe affected. Cell death occurs in tissue surrounding the infarct uponreperfusion of blood to the area; thus, the compositions are effectiveif administered at the onset of the infarct, during reperfusion, orshortly thereafter. The present invention includes methods of treatingreperfusion damage by administering a therapeutically effective amountof the compounds having agonist activity on an LPA receptor to a patientin need of such therapy.

The invention further encompasses a method of reducing the damageassociated with myocardial and cerebral infarctions for patients with ahigh risk of heart attack and stroke by administering a therapeuticallyeffective amount of the compounds having agonist activity on an LPAreceptor to a patient in need of such therapy. Preferably, treatment ofsuch damage is by parenteral administration of such compounds. Any othersuitable method can be used, however, for instance, direct cardiacinjection in the case of myocardial infarct. Devices for such injectionare known in the art, for instance the Aboject cardiac syringe.

The invention further provides methods of limiting and preventingapoptosis in cells, or otherwise preserving cells, during the culture ormaintenance of mammalian organs, tissues, and cells, by the addition ofan effective amount of the compounds having agonist activity on an LPAreceptor to any media or solutions used in the art of culturing ormaintaining mammalian organs, tissues, and cells.

The invention further encompasses media and solutions known in the artof culturing and maintaining mammalian organs, tissues and cells, whichinclude an amount of the compounds having agonist activity on an LPAreceptor which is effective to preserve or restore cell, tissue or organfunction, or limit or prevent apoptosis of the cells in culture. Theseaspects of the invention encompass mammalian cell culture mediaincluding an effective amount of at least one compounds having agonistactivity on an LPA receptor and the use of such media to preserve orrestore cell, tissue or organ function, or to limit or prevent apoptosisin mammalian cell culture. An effective amount is one which decreasesthe rate of apoptosis and/or preserves the cells, tissue or organ. Suchcompounds can limit or prevent apoptosis under circumstances in whichcells are subjected to mild traumas which would normally stimulateapoptosis. Exemplary traumas can include, but are not limited to, lowlevel irradiation, thawing of frozen cell stocks, rapid changes in thetemperature, pH, osmolarity, or ion concentration of culture media,prolonged exposure to non-optimal temperature, pH, osmolarity, or ionconcentration of the culture media, exposure to cytotoxins,disassociation of cells from an intact tissue in the preparation ofprimary cell cultures, and serum deprivation (or growth in serum-freemedia).

Thus, the invention encompasses compositions comprising tissue culturemedium and an effective amount of the compounds having agonist activityon an LPA receptor. Serum-free media to which the compositions can beadded as anti-apoptotic media supplements include, but are not limitedto, AIM VP Media, Neuman and Tytell's Serumless Media, Trowell's T8Media, Waymouth's MB 752/1 and 705/1 Media, and Williams' Media E. Inaddition to serum-free media, suitable mammalian cell culture media towhich the compounds having agonist activity on an LPA receptor can beadded as anti-apoptotic media supplements include, but are not limitedto, Basal Media Eagle's, Fischer's Media, McCoy's Media, Media 199, RPMIMedia 1630 and 1640, Media based on F-10 & F-12 Nutrient Mixtures,Leibovitz's L-15 Media, Glasgow Minimum Essential Media, and Dulbecco'sModified Eagle Media. Mammalian cell culture media to which thecompounds having agonist activity on an LPA receptor can be addedfurther include any media supplement known in the art. Exemplarysupplements include, but are not limited to, sugars, vitamins, hormones,metalloproteins, antibiotics, antimycotics, growth factors,lipoproteins, and sera.

The invention further encompasses solutions for maintaining mammalianorgans prior to transplantation, which solutions include an effectiveamount of the compounds having agonist activity on an LPA receptor, andthe use of such solutions to preserve or restore organ function or tolimit or prevent apoptosis in treated mammalian organs during theirsurgical removal and handling prior to transplantation. The solutionscan be used to rush, perfuse and/or store the organs. In all cases,concentrations of the compounds (having agonist activity on an LPAreceptor) required to limit or prevent damage to the organs can bedetermined empirically by one skilled in the art by methods known in theart.

In addition to the foregoing, the compounds having agonist activity onan LPA receptor can be topically applied to the skin to treat a varietyof dermatologic conditions. These conditions include, but are notlimited to, hair loss and wrinkling due to age and/or photo damage. Thepresent invention also encompasses, therefore, methods of treatingdermatological conditions. In particular, hair loss can be caused byapoptosis of the cells of the hair follicles (Stenn et al., 1994).Therefore, the compounds having agonist activity on an LPA receptor aresuitable for use in topical treatment of the skin to prevent continuedhair loss.

The various dermatologic conditions are preferably treated by topicalapplication of an effective amount of a compound having agonist activityon an LPA receptor (or compositions which contain them). An effectiveamount of such compounds is one which ameliorates or diminishes thesymptoms of the dermatologic conditions. Preferably, the treatmentresults in resolution of the dermatologic condition or restoration ofnormal skin function; however, any amelioration or lessening of symptomsis encompassed by the invention.

EXAMPLES

The following examples are intended to illustrate, but by no means areintended to limit, the scope of the present invention as set forth inthe appended claims.

General Methods

All reagents were purchased from Sigma-Aldrich Chemical Co., FisherScientific (Pittsburgh, Pa.), Bedukian Research (Danbury, Conn.) andToronto Research Chemicals (North York, ON, Canada) and were usedwithout further purification. Phosphonate analogs were purchased fromLancaster (Pelham, N.H.; n-decyl-phosphonate (9a)), PolyCarbon (Devens,Mass.; n-dodecyl-phosphonate (9b)), Alfa Aesar (Ward Hill, Mass.;n-tetradecyl-phosphonate (9c) and n-octadecyl-phosphonate (9d)). LPA18:1, DGPP, Ser-PA, and Tyr-PA were obtained from Avanti Polar Lipids(Alabaster, Ala.). Melting points were determined on a Thomas-Hoovercapillary melting point apparatus and are uncorrected. Routinethin-layer chromatography (TLC) was performed on 250 μM glassbackedUNIPLATES (Analtech, Newark, Del.). Flash chromatography was performedon pre-packed silica gel columns using a Horizon HPFC system (Biotage,Charlottesville, Va.). ¹H and ³1P NMR spectra were obtained on a BrukerAX 300 (Billerica, Mass.) spectrometer. Chemical shifts for ¹H NMR arereported as parts per million (ppm) relative to TMS. Chemical shifts for³1P NMR are reported as parts per million (ppm) relative to 0.0485 Mtriphenylphosphate in CDCl₃. Mass spectral data was collected on aBruker ESQUIRE electrospray/ion trap instrument in the positive andnegative ion modes. Elemental analyses were performed by AtlanticMicrolab Inc., Norcross, Ga.

Example 1 Synthesis of Phosphoric Acid di-tert-butyl Ester AlkenylEsters (4a-f)

Commercially available unsaturated fatty alcohols (3a-f) were used asstarting materials. To a stirred solution of alcohol (2.5 mmol) anddi-tert-butyl-N,N-diisopropyl phosphoramidite (1.51 g, 4 mmol) inmethylene chloride (60 mL) was added 1H-tetrazole (578 mg, 8.25 mmol).After 30 minutes of stirring the mixture was cooled to 0° C. and 0.3 mLof 50% hydrogen peroxide was added. The mixture was stirred for 1 h.,diluted with methylene chloride (100 mL), washed with 10% sodiummetabisulfite (2×50 ml), saturated sodium bicarbonate (2×50 ml), water(50 ml), and brine (50 ml). The organic layer was dried over anhydroussodium sulfate, filtered, and concentrated under vacuum. The resultingcrude products were purified by silica gel chromatography usinghexane/ethyl acetate (7:3) to elute the desired products, di-t-Bocprotected fatty alcohol phosphates (4a-f).

Phosphoric acid di-tert-butyl ester dec-9-enyl ester (4a): Isolated asclear oil (75% yield). ¹H NMR (CDCl₃): δ5.80 (m, 1H), 4.95 (m, 2H), 3.95(q, J=7.5 Hz, 2H), 2.03 (q, J=7.1 Hz, 2H), 1.65 (quintet, 2H), 1.48 (s,18H), 1.30 (br s, 10H); ³1P NMR (CDCl₃): δ7.90; MS: [M+²³Na] at m/z371.3.

Phosphoric acid di-tert-butyl ester dec-4-enyl ester (4b): Isolated asclear oil (68% yield). ¹H NMR (CDCl₃): δ5.25 (m, 2H), 3.84 (q, J=6.8 Hz,2H), 2.05 (q, J=7.0 Hz, 2H), 1.98 (q, J=6.8 Hz, 2H), 1.61 (quintet, 2H),1.42 (s, 18H), 1.22 (br s, 6H), 0.80 (t, J=7.2 Hz, 3H); ³1P NMR(MeOH-d₄): δ7.90; MS: [M+²³ Na] at m/z 371.3.

Phosphoric acid di-tert-butyl ester dodec-9-enyl ester (4c): Isolated asclear oil (70% yield). ¹H NMR (CDCl₃): δ5.26 (m, 2H), 3.88 (q, J=6.6 Hz,2H), 1.94 (m, 4H), 1.59 (quintet, 2H), 1.42 (s, 18H), 1.24 (br s, 10H),0.89 (t, J=7.5 Hz, 3H); ³1P NMR (CDCl₃): δ7.80; MS: [M+²³Na] at m/z399.5.

Phosphoric acid di-tert-butyl ester tetradec-9-enyl ester (4d): Isolatedas clear oil (68% yield). ¹H NMR (CDCl₃): δ5.34 (t, J=5.2 Hz, 2H), 3.94(q, J=6.6 Hz, 2H), 2.01 (m, 4H), 1.65 (quintet, 2H), 1.48 (s, 18H), 1.30(br s, 18H), 0.90 (t, J=7.4 Hz, 3H); ³1P NMR (CDCl₃): δ7.90; MS: [M+²³Na] at m/z 427.4.

Phosphoric acid di-tert-butyl ester tetradec-11-enyl ester (4e):Isolated as clear oil (82% yield). ¹H NMR (CDCl₃): δ5.34 (m, 2H), 3.94(q, J=6.5 Hz, 2H), 2.01 (m, 4H), 1.65 (quintet, 2H), 1.48 (s, 18H), 1.23(br s, 14H), 0.95 (t, J=7.4 Hz, 3H); ³1P NMR (CDCl₃): δ8.10; MS:[M+²³Na] at m/z 427.4.

Phosphoric acid di-tert-butyl ester octadec-9-enyl ester (4f): Isolatedas clear oil (72% yield). ¹H NMR (CDCl₃): δ5.34 (m, 2H), 3.94 (q, J=6.9Hz, 2H), 2.01 (m, 4H), 1.66 (quintet, 2H), 1.48 (s, 18H), 1.28 (br s,22H), 0.88 (t, J=6.6 Hz, 3H); ³1P NMR (CDCl₃): δ8.10; MS: [M+²³ Na] atm/z 483.5.

Example 2 Synthesis of Phosphoric Acid Mono Alkenyl Esters (5a-f)

The Boc-protected FAPs (4a-f) were deprotected with TFA to yield thecorresponding unsaturated FAPs (5a-f). To a solution of 100 mg of 1a-6ain methylene chloride (20 mL), trifluoroacetic acid (0.3 mL) was added.The mixture was allowed to stir for 4 h., and TLC showed the completionof the reaction. Solvents were evaporated; the residue was washed withmethylene chloride (2×20 mL), and concentrated under vacuum to yield thedesired phosphoric acid mono alkenyl esters as colorless oils.

Phosphoric acid monodec-9-enyl ester (5a): Isolated as an oil (85%). ¹HNMR (MeOH-d₄): δ5.74 (m, 1H), 4.88 (m, 2H), 3.90 (q, J=6.6 Hz, 2H), 2.01(q, J=6.9 Hz, 2H), 1.61 (quintet, 2H), 1.28 (br s, 10H); ³1PNMR(MeOH-d₄): δ17.84; MS: [M-H]— at m/z 235.2. Anal. (C₁₀H₂₁O₄P.0.1H₂O)C,H.

Phosphoric acid monodec-4-enyl ester (5b): Isolated as an oil (78%). ¹HNMR (MeOH-d₄): δ5.31 (m, 2H), 3.84 (q, J=6.8 Hz, 2H), 2.05 (q, J=7.0 Hz,2H), 1.98 (q, J=6.8 Hz, 2H), 1.61 (quintet, 2H), 1.22 (br s, 6H), 0.80(t, J=7.2 Hz, 3H); ³1P NMR (MeOH-d₄): δ17.45; MS: [M-H]— at m/z 235.2.Anal. (C₁₀H₂₁O₄P.0.5H₂O)C, H.

Phosphoric acid monododec-9-enyl ester (5c): Isolated as an oil (82%).¹H NMR (DMSO/MeOH-d₄): δ5.28 (m, 2H), 3.82 (q, J=6.6 Hz, 2H), 1.96 (m,4H), 1.54 (m, 2H), 1.25 (br s, 10H), 0.88 (t, J=7.2 Hz, 3H); ³1P NMR(MeOH-d₄): Δ 16.22; MS: [M-H]— at m/z 263.0. Anal. (C₁₂H₂₅O₄P.0.6H₂O)C,H.

Phosphoric acid monotetradec-9-enyl ester (5d): Isolated as an oil(84%). ¹H NMR (CDCl₃/MeOH-d₄): δ5.21 (m, 2H), 3.84 (q, J=6.5 Hz, 2H),1.91 (m, 4H), 1.54 (m, 2H), 1.20 (br s, 14H), 0.78 (m, 3H); ³1P NMR(MeOH-d₄): Δ 16.20; MS: [M-H]— at m/z 291.4. Anal. (C₁₄H₂₉O₄P.0.25H₂O)C,H.

Phosphoric acid monotetradec-11-enyl ester (5e): Isolated as an oil(78%). ¹H NMR (MeOH-d₄): δ5.24 (m, 2H), 3.88 (q, J=6.6 Hz, 2H), 1.95 (m,4H), 1.58 (m, 2H), 1.25 (br s, 14H), 0.86 (t, J=7.1 Hz, 3H); ³1P NMR(MeOH-d₄): δ16.20; MS: [M-H]— at m/z 291.3. Anal. (C₁₄H₂₉O₄P)C, H.

Phosphoric acid monooctadec-9-enyl ester (5f): Isolated as an oil (86%).¹H NMR (MeOH-d₄): δ5.30 (m, 2H), 3.91 (q, J=6.6 Hz, 2H), 2.00 (m, 4H),1.62 (quintet, 2H), 1.26 (br s, 22H), 0.86 (t, J=6.0 Hz, 3H); ³1P NMR(MeOH-d₄): δ16.21; MS: [M-H]— at m/z 347.4. Anal. (C₁₈H₃₇O₄P.0.4H₂O)C,H.

Example 3 Synthesis of Thiophosphoric Acid O,O′-bis-(2-cyano-ethyl)Ester O″-alkyl/alkenyl Esters (7a-g)

Commercially available saturated or unsaturated fatty alcohols (6a-g)were used as starting materials. A solution of alcohol (2.0 mmol),bis-(2-cyanoethyl)-N,N-diisopropyl phosphoramidite (1.085 g, 4 mmol) and1H-tetrazole (420 mg, 6 mmol) was stirred for 30 minutes at roomtemperature, followed by the addition of elemental sulfur (200 mg) andthe mixture was refluxed for 2 h. The reaction mixture was cooled toroom temperature and solvents were evaporated under vacuum. Addition ofethyl acetate (30 mL) precipitated excess sulfur, which was filteredout, and the solvent was evaporated to give the crude mixture. Themixture was purified by flash chromatography to give the desiredproducts as colorless oils.

Thiophosphoric acid O,O′-bis-(2-cyano-ethyl) ester O″-decyl ester (7a):Isolated as colorless oil (72% yield). ¹H NMR (CDCl₃): δ4.21-4.35 (m,4H), 4.12 (m, 2H), 2.8 (t, J=6.3 Hz, 4H), 1.68 (quintet, 2H), 1.26 (brs, 14H), 0.88 (t, J=6.0 Hz, 3H); MS: [M+²³Na] at m/z 383.4.

Thiophosphoric acid O,O′-bis-(2-cyano-ethyl) ester O″-dodecyl ester(7b): Isolated as colorless oil (84% yield). ¹H NMR (CDCl₃): δ4.26-4.33(m, 4H), 4.12 (m, 2H), 2.8 (t, J=6.2 Hz, 4H), 1.71 (quintet, 2H), 1.26(br s, 14H), 0.88 (t, J=6.6 Hz, 3H); MS: [M+²³Na] at m/z 411.4.

Thiophosphoric acid O,O′-bis-(2-cyano-ethyl) ester O″-tetradecyl ester(7c): Isolated as clear oil (82% yield). ¹H NMR (CDCl₃): δ4.25-4.33 (m,4H), 4.12 (m, 2H), 2.8 (t, J=6.0 Hz, 4H), 1.71 (quintet, 2H), 1.26 (brs, 18H), 0.88 (t, J=6.6 Hz, 3H); MS: [M+²³Na] at m/z 439.5.

Thiophosphoric acid O,O′-bis-(2-cyano-ethyl) ester O″-dec-9-enyl ester(7d): Isolated as clear oil (76% yield). ¹H NMR (CDCl₃): δ5.81 (m, 1H),4.96 (m, 2H), 4.22-4.32 (m, 4H), 4.11 (m, 2H), 2.8 (t, J=6.3 Hz, 4H),2.01 (t, J=6.6 Hz, 4H), 1.70 (quintet, 2H), 1.31 (br s, 10H); MS:[M+²³Na] at m/z 381.3.

Thiophosphoric acid O,O′-bis-(2-cyano-ethyl) ester O″-dodec-9-enyl ester(7e): Isolated as clear oil (80% yield). ¹H NMR (CDCl₃): δ5.34 (m, 2H),4.25-4.33 (m, 4H), 4.11 (m, 2H), 2.8 (t, J=6.0 Hz, 4H), 2.07 (m, 2H),1.70 (quintet, 2H), 1.31 (br s, 10H), 0.96 (t, J=7.5 Hz, 3H); MS:[M+²³Na) at m/z 409.5.

Thiophosphoric acid O,O′-bis-(2-cyano-ethyl) ester O″-tetradec-9-enylester (7f): Isolated as clear oil (75% yield). ¹H NMR (CDCl₃): δ5.35 (m,2H), 4.25-4.33 (m, H), 4.12 (m, 2H), 2.78 (t, J=6.0 Hz, 4H), 2.02 (m,2H), 1.71 (quintet, 2H), 1.31 (br s, 14H), 0.90 (t, J=7.2 Hz, 3H); MS:[M+²³Na] at m/z 437.5.

Thiophosphoric acid O,O′-bis-(2-cyano-ethyl) ester O″-octadec-9-enylester (7 g): Isolated as clear oil (72% yield). ¹H NMR (CDCl₃): δ5.35(m, 2H), 4.27-4.31 (m, 4H), 4.12 (m, 2H), 2.78 (t, J=6.0 Hz, 4H), 2.02(m, 2H), 1.71 (quintet, 2H), 1.27 (br s, 22H), 0.88 (t, J=7.2 Hz, 3H);MS: [M+²³ Na] at m/z 493.5.

Example 4 Synthesis of Thiophosphoric Acid O-alkyl/alkenyl Esters (8a-g)

Thiophosphoric acid O,O′-bis-(2-cyano-ethyl) ester O″-alkyl/alkenylesters (7a-7 g) were used as starting materials. A solution of 100 mg of7a-7 g in methanolic KOH (10 mL) was stirred for 2 h., and TLC showedthe completion of the reaction. The solvent was evaporated to give thecrude product, which was dissolved in water (20 mL), and acidified withHCl. The aqueous mixture was extracted with ethyl acetate (2×50 mL),organic layer was dried over sodium sulfate and concentrated undervacuum to give the desired compound as light yellow colored oil.

Thiophosphoric acid O-decyl ester (8a): Isolated as light yellow coloredoil (80% yield). ¹H NMR (DMSO): δ3.86 (m, 2H), 1.56 (quintet, 2H), 1.24(br s, 14H), 0.86 (t, J=6.0 Hz, 3H); MS: [M-H]— at m/z 253.2. Anal.(C₁₀H₂₃O₃PS)C, H.

Thiophosphoric acid O-dodecyl ester (8b): Isolated as light yellowcolored oil (73% yield). ¹H NMR (DMSO): δ3.84 (m, 2H), 1.56 (quintet,2H), 1.24 (br s, 18H), 0.83 (t, J=6.9 Hz, 3H); MS: [M-H]— at m/z 280.9.Anal. (C₁₂H₂₇O₃PS.0.5H₂O)C, H

Thiophosphoric acid O-tetradecyl ester (8c): Isolated as light yellowcolored oil (70% yield). ¹H NMR (DMSO): δ3.85 (m, 2H), 1.56 (quintet,2H), 1.24 (br s, 22H), 0.85 (t, J=6.0 Hz, 3H); MS: [M-H]— at m/z 309.4.Anal. (C₁₄H₃₁O₃PS.0.25H₂O)C, H.

Thiophosphoric acid O-dec-9-enyl ester (8d): Isolated as light yellowcolored oil (76% yield). ¹H NMR (DMSO): δ5.79 (m, 1H), 4.94 (m, 2H),3.85 (m, 2H), 2.01 (q, J=6.6 Hz, 4H), 1.55 (quintet, 2H), 1.26 (br s,10H); MS: [M-H]— at m/z 251.1. Anal. (C₁₀H₂₁O₃PS)C, H.

Thiophosphoric acid O-dodec-9-enyl ester (8e): Isolated as light yellowcolored oil (80% yield). ¹H NMR (DMSO): Δ 5.31 (m, 2H), 3.85 (q, J=6.6Hz, 2H), 1.99 (m, 4H), 1.56 (quintet, 2H), 1.26 (br s, 10H), 0.91 (t,J=7.5 Hz, 3H); MS: [M-H]— at m/z 279.5. Anal. (C₁₂H₂₅O₃PS0.35H₂O)C, H.

Thiophosphoric acid O-tetradec-9-enyl ester (8f): Isolated as lightyellow colored oil (72% yield). ¹H NMR (DMSO): δ5.32 (m, 2H), 3.85 (m,2H), 1.98 (m, 4H), 1.55 (quintet, 2H), 1.26 (br s, 14H), 0.86 (t, J=6.9Hz, 3H); MS: [M-H]— at m/z 307.5. Anal. (C₁₄H₂₉O₃PS.0.3H₂O)C, H.

Thiophosphoric acid O-octadec-9-enyl ester (8 g): Isolated as lightyellow colored oil (82% yield). ¹H NMR (DMSO): δ5.32 (m, 2H), 3.85 (m,2H), 1.97 (m, 4H), 1.55 (quintet, 2H), 1.24 (br s, 22H), 0.85 (t, J=6.9Hz, 3H); MS: [M-H]— at m/z 363.5. Anal. (C₁₈H₃₇O₃PS.0.3H₂O)C, H.

Example 5 Synthesis of (1,1-Difluoro-pentadecyl) Phosphonic Acid DiethylEster (10)

To a solution of diethyl difluoromethanephosphonate (1.0 g, 5.316 mmol)in THF (50 mL) 2 M LDA (626 mg, 5.847 mmol) was added at −78° C. andstirred for 30 min. Tetradecyl bromide (1.474 g, 5.316 mmol) in THF (10mL) was added to the mixture at −78° C. and the reaction mixture wasstirred overnight. THF was evaporated and the residual oil was purifiedby flash chromatography using 30% ethyl acetate in hexane as eluent togive 817 mg (40%) of compound 10 as colorless oil. ¹H NMR (CDCl₃): δ4.26(m, 4H), 2.05 (m, 2H), 1.56 (m, 2H), 1.37 (t, J=6.9 Hz, 6H), 1.25 (br s,22H), 0.87 (t, J=6.6 Hz, 3H); MS: [M+²³Na] at m/z 407.2.

Example 6 Synthesis of (1,1-Difluoro-pentadecyl) Phosphonic Acid (11)

To a solution of vacuum dried 10 (225 mg, 0.585 mmol) in methylenechloride (5 mL) bromotrimethyl silane (895 mg, 5.85 mmol) was added andthe mixture was stirred at room temperature. TLC showed completion ofthe reaction after 6 h. Solvents were removed under reduced pressure,and the residue was stirred in 95% methanol (3 mL) for 1 h. The mixturewas concentrated under reduced pressure, dried under vacuum to give 150mg (78%) of 11 as light yellow solid. mp 66-69° C.; ₁H NMR (CD₃OD):δ2.03 (m, 2H), 1.59 (m, 2H), 1.24 (br s, 22H), 0.90 (t, J=6.6 Hz, 3H);MS: [M-H]— at m/z 327.3. Anal. (C₁₅H₃]F₂O₃P.0.2H₂O)C, H.

Example 7 Analysis of Compounds for LPA Receptor Agonist or AntagonistActivity

Compounds were tested for their ability to induce or inhibit LPA-inducedcalcium transients in RH7777 rat hepatoma cells stably expressing LPA₁,LPA₂, and LPA₃ receptors and in PC-3 that express LPA₁₋₃ endogenously,using a FlexStation II automated fluorometer (Molecular Devices,Sunnyvale, Calif.) (Fischer et al., 2001; Virag et al., 2003).

RH7777 cells stably expressing either LPA₁, LPA₂ or LPA₃ (Fischer et al2001; Virag et al., 2003) or PC-3 cells were plated on poly-Dlysine-coated black wall clear bottom 96-well plates (Becton Dickinson,San Jose, Calif.) with a density of 50000 cells/well, and culturedovernight. The culture medium (DMEM containing 10% FBS) was thenreplaced with modified Krebs solution (120 mM NaCl, 5 mM KCl, 0.62 mMMgSO₄, 1.8 mM CaCl₂, 10 mM HEPES, 6 mM glucose, pH 7.4) and the cellswere serum starved for 6-8 hours (12 h for PC-3 cells). Cells wereloaded with Fura-2 AM for 35 minutes in modified Krebs medium. TheFura-2 was removed before loading the plate in the FlexStationinstrument by replacing the medium once again with 100 μl modified Krebsmedium/well. Plates were incubated for 4 minutes in the instrument toallow for warming to 37° C. Changes in intracellular Ca²⁺ concentrationwere monitored by measuring the ratio of emitted light intensity at 520nm in response to excitation by 340 nm and 380 nm wavelength lights,respectively. Each well was monitored for 80-120 seconds. 50 μl of thetest compound (3× stock solution in modified Krebs) was addedautomatically to each well 15 seconds after the start of themeasurement. Time courses were recorded using the SoftMax Pro software(Molecular Devices, Sunnyvale, Calif.). Ca⁺ transients were quantifiedautomatically by calculating the difference between maximum and baselineratio values for each well.

Selected compounds were tested for PPARγ activation in CV1 cells,transfected with an acyl-coenzyme A oxidase-luciferase (PPRE-Acox-Rluc)reporter gene construct as previously reported (Zhang et al., 2004). Theassay of PPARγ activation in CV1 cells was run as reported in Zhang etal. Briefly, CV-1 cells were plated in 96-well plates (5×10₃ cells perwell) in Dulbecco's modified Eagle's medium supplemented with 10% fetalbovine serum. The next day, the cells were transiently transfected with125 ng of pGL3-PPRE-Acox-Rluc, 62.5 ng of pcDNAI-PPARγ, and 12.5 ng ofpSV-β-galactosidase (Promega, Madison, Wis.) using LipofectAMINE 2000(Invitrogen). Twenty-four hours after System (Promega) and theGalacto-Light Plus™ System (Applied Biosystems, Foster City, Calif.),respectively, samples were run in quadruplicate and the mean ±standarderrors were calculated. Data are representative of at least twoindependent transfections. Student's t-test was used for null hypothesistesting and P<0.05 was considered transfection, cells were treated with1% FBS supplemented OptiMEMI (Invitrogen) containing DMSO or 10 μM testcompound dissolved in DMSO for 20 h. Luciferase and β-galactosidaseactivities were measured with the Steady-Glo® Luciferase Assay assignificant (in the figures P<0.05 is denoted by * and P<0.01 is **).

According to the original two-point contact model (Wang et al., 2001;Sardar et al., 2002), both a polar phosphate head group and ahydrophobic tail are required for specific interactions with the LPAGPCRs (Fischer et al., 2001). The phosphate group was identified as anecessary component that interacts with two positively charged conservedamino acid residues in the third and seventh transmembrane helices ofthe LPA receptors (Wang et al., 2001). The hydrophobic tail interactswith a pocket of hydrophobic residues in the transmembrane regions ofthe receptors, significantly contributing to the ligand-receptor binding(Wang et al., 2001; Sardar et al., 2002).

Based on this model the inventors identified DGPP and dioctylphosphatidic acid as selective LPA₁ and LPA₃ antagonists and FAPs assubtype selective agonists/antagonists of LPA₁₋₃ receptors (Fischer etal., 2001; Virag et al., 2003). Bandoh et al. (2000) showed that LPA₃prefers unsaturated fatty acyl LPA species over saturated LPAs.Replacement of the phosphate with a phosphonate renders compoundsmetabolically stable against degradation by lipid phosphatephosphatases. Phosphonate modification also affects ligand-receptorinteractions by reducing charge density on the polar head group.Phosphonate analogs of LPA have been studied recently and are lesspotent than LPA (Hooks et al., 2001; Xu et al., 2002). Alternatively,thiophosphate in place of phosphate yielded metabolically stablecompounds with increased charge on the polar head group such as OMPT, aselective LPA₃ agonist (Hasegawa et al., 2003; Qian et al., 2003).

To explore the effects of these modifications along with the variationsin the side chain in the FAP structure, the inventors synthesized aseries of FAP analogs with an unsaturation at different positions in thesidechain (5a-f), thiophosphates (8a-g) and phosphonates (9ad, 11).These new analogs were evaluated as agonists and antagonists withrespect to LPA₁₋₃. Saturated FAP analogs containing 10, 12 or 14 carbons(2a-c) were previously shown to be the most effective agonists and/orinhibitors at LPA₁₋₃ in the initial study (Virag et al., 2003). For thisreason the inventors synthesized and characterized modified FAP analogswith these optimum chain lengths.

Each FAP analog was tested for the ability to induce Ca²⁺ transients inRH7777 cells transfected with LPA₁₋₃ receptors (agonism), as well as theability to inhibit LPA-induced Ca²⁺ transients in the same cells(antagonism) (see Table 3). None of the compounds examined in this studyinduced intracellular Ca²⁺ transients when applied up to a concentrationof 30 μM in non-transfected RH7777 cells. The effects of unsaturation atdifferent positions, modification of head group by phosphonate, difluorophosphonate and thiophosphate with/without unsaturation on the activityof C-14 analogs at LPA₁₋₃ receptors are shown in FIG. 2. Thesemodifications dramatically changed the pharmacological properties ofFAPs on LPA₁₋₃ receptors. The mono-unsaturated FAP analogs (5a-e) showeda trend of increasing the potency and/or efficacy when compared to thesaturated analogs, except C-10 analogs, without changing their ligandproperties as agonists or antagonists at the LPA₂ and LPA₃ receptors(Table 3). The position of the double bond also had an impact on theactivity. Comparison of the activities between decenyl regio isomers 5aand 5b, suggests that the C₉═C₁₀ double bond, as found in LPA 18:1, waspreferred over C₄═C₅ in activating LPA₂ receptor (EC₅₀=3800 nM for 5aversus >10000 nM for 5b). Though 5b (K_(i)=370 nM) was moderately moreactive than 5a (K_(i)=504 nM), the preference for the double bondposition was much less pronounced for inhibition of LPA₃ receptor.

Similarly, the LPA₂ receptor showed preference for C₉═C₁₀ unsaturationbetween the tetradecenyl isomers 5d (EC₅₀=397 nM) and 5e (EC₅₀=4100 nM),and LPA₃ showed no significant preference for double bond position. Incontrast, LPA₁ preferred C₁₁═C₁₂ over C₉═C₁₀ between 5d (K_(i)=1146 nM)and 5e (K_(i)=457 nM), indicating the possibility of a differentialconformational requirement in the side chain for each of the three LPAreceptors (FIG. 2). In the unsaturated series, only tetradecenylcompounds (5d, 5e) antagonized the LPA response at LPA₁ receptor. Thisfurther supports our belief that the length of the side chain iscritical for interaction with LPA receptors.

The replacement of phosphate with a thiophosphate as the headgroup in10-, 12-, and 14-carbon saturated FAP analogs (8a-c) had a major impacton their agonist/antagonist properties at all three LPA receptorsubtypes. At LPA₁, the thiophosphate modification completely abolishedthe inhibitory effects of the original FAP analogs. At LPA₂ on the otherhand, the thiophosphate invariably increased the efficacy of theoriginal FAP to 100%. At the LPA₃ receptor, the saturated thiophosphateFAP analogs consistently showed improved inhibition of the LPA responsecompared to the original FAPs. Dodecyl-thiophosphate (8b) is the mostpotent agonist and antagonist in the saturated thiophosphate analogs atLPA₂ (EC₅₀=1000 nM) and LPA₃ (K_(i)=14 nM), respectively. These resultsare consistent with our two-point contact model as the increase in thecharge density, influenced by the properties of the hydrophobic tail,increased the agonist or antagonist properties of the FAP.

Next, we investigated the effect of combining a thiophosphate headgroupwith mono-unsaturation (C₉═C₁₀) in the side chain. The combination ofthe thiophosphate headgroup with C₉═C₁₀ unsaturation resulted in analogs(8d-8f) with agonist/antagonist properties that were the combinedproperties of the saturated thiophosphates and unsaturated FAPssubstantially lowering the EC₅₀ and IC₅₀ values. Similar to thesaturated thio analogs, compounds 8d-8f were inactive at LPA₁ receptor.When the effects of saturated and unsaturated C₁₂, C₁₄ thiophosphates atLPA₂ and LPA₃ are compared, there is an increase in potency with theunsaturated analogs at LPA₂ with a minimal change in the potency at LPA₃receptor. The tetradec-9-enyl thiophosphate (8f) compound needs to bediscussed separately. It has retained the features of the saturated thioanalogs at LPA₁, as it had no effect on the LPA-induced Ca²⁺mobilization. On the other hand, at 8f was found to be the best agonistat LPA₂ (EC₅₀=480 nM) and most potent antagonist at LPA₃ (K₁=14 nM)among all C-10, -12, and -14 thiophosphate analogs (FIGS. 2B and 2D).Dodec-9-enyl analog (8e) was an equipotent antagonist as 8f at the LPA₃receptor. These differences in the effects of the thiophosphate analogsat the LPA receptor subtypes may provide us with a practical advantagein developing future subtype-selective agonists and antagonists, asshort-chain thiophosphates interact selectively with LPA₂ and LPA₃receptors.

Oleoyl-phosphate (5f), an unsaturated FAP analog of oleoly-LPA, did notinhibit nor did it activate Ca²⁺ mobilization in cells expressingLPA₁₋₃. However, it potentiated LPA response at all three LPA receptorswhen the two compounds were co-applied. This observation led us to thehypothesis that by increasing the charge density of the 5f headgroup byreplacing the phosphate with a thiophosphate, we may increase thebinding of this compound to the receptors that is essential to turn thisanalog into an agonist. To test this hypothesis, we synthesized andevaluated the oleoylthiophosphate (8 g) at LPA₁₋₃ receptors. Inagreement with our prediction, compound 8g was a partial agonist at LPA₁(EC₅₀ (E_(max))=193 nM (80%)), and LPA₃ (EC₅₀ (E_(max))=546 nM (78%)),and a potent and full agonist at LPA₂ with the EC₅₀ of 244 nM(E_(max)=175% of LPA response), lower than that of oleoyl-LPA (EC₅₀=300nM). The dose responses of 8 g, comparing its effects with LPA 18:1 atLPA₁₋₃ receptors, are shown in FIG. 3.

The phosphonate analogs (9a-d) were weaker inhibitors and agonists atthe LPA receptors than their phosphate counterparts, consistent withdata reported previously (Hooks et al., “Lysophosphatidic Acid-InducedMitogenesis Is Regulated by Lipid Phosphate Phosphatases and IsEdg-Receptor Independent,” J. Biol. Chem. 276:4611-4621 (2001)).However, tetradecyl-phosphonate (9c) inhibited LPA-induced Ca²⁺mobilization at all three receptor subtypes with IC₅₀ values in themicromolar range, thus becoming the first pan antagonist of the EDGfamily LPA receptors (FIG. 2). The importance of this finding istwofold. Compound 9c is the only known inhibitor of the LPA₂ receptorsubtype apart from Ki16425 that exerts only a modest and partialinhibition (Ohta et al., 2003). Compound 9c, with a simpler structureand phosphonate headgroup, is presumably resistant to degradation bylipid phosphate phosphatases. These features make this molecule a goodlead structure for further development of pan-antagonists for the LPA₁₋₃receptors. We synthesized compound II, a difluorophosphonate analog ofcompound 9c, with an isosteric replacement of phosphonate bydifluorophosphonate and tested at LPA₁₋₃ receptors. This compoundretains the metabolic stability against phosphatases and at the sametime increases the acidity of the phosphonate group, which presumablyincreases the binding to the receptor. Increase in the acidity ofphosphonate group by the two fluorine atoms in compound II reversed thecompound from an antagonist to a weak and partial agonist with an EC₅₀of 10 μM (E_(max)=40%) at LPA₂ receptor. Compound II showed improvedantagonistic activity at LPA₃ (K_(i)=575 nM) compared to 9c (K_(i)=1120nM), while it showed partial antagonism (≈K_(i)=788 nM; 40% inhibitionof LPA response) at LPA₁.

LPA was shown to activate mitogenic and motogenic signaling in PC-3cells (Kue et al., 2002). RT-PCR analysis of PC-3 cells, anandrogen-independent human prostate cancer cell line, showed expressionof transcripts encoding all three LPA receptors (Daaka et al., 2002). Wetested the FAP analogs in PC-3 cells, which unlike the transfectedRH7777 cells endogenously express LPA₁₋₃ receptors. Since PC-3 cellsexpress LPA₁₋₃ receptors, the effects shown by the FAP compounds (Table3) represent the combination of the effects of these compounds at thethree LPA receptors. These experiments confirmed the pharmacologicalproperties of the FAP analogs obtained from RH7777 cells expressing eachLPA receptor individually. Thiophosphate analogs (8e and 8f) showed bothindependent activation and inhibition of LPA-induced Ca²⁺ transients inPC-3 cells as they have different effects at each of the LPA₁₋₃receptors. Oleoyl-thiophosphate (8 g) showed a maximal response of 30%of maximal LPA response, with no inhibition of LPA response, consistentwith data from transfected RH7777 cells. Similarly the inhibitoryactivity shown by other compounds (Table 3) is a combination of effectsof these compounds at individual LPA₁₋₃ receptors. The consistency ofthe results obtained from PC-3 cells that endogenously express LPAreceptors with those results obtained using transfected RH7777 cellsvalidates our assay systems.

To compare the effects of these FAP analogs at LPA receptors with theother available agonists and antagonists, we tested DGPP 8:0, Ki16425,N-acyl serine phosphoric acid (Ser-PA), N-acyl tyrosine phosphoric acid(Tyr-PA), and VPC12249 in our RH7777 cell system. This comparison, wherea single test system is used for all compounds, has the benefit ofproviding us with reliable information on the relative effectiveness ofthese compounds despite the inherent shortcomings the individual testsystems may have. Our results were consistent with previously publisheddata for DGPP 8:0, Ser-PA and Ki16425, however we encountereddifferences for Tyr-PA, and VPC12249 (Table 3). DGPP 8:0 was identifiedin our lab as a subtype-selective inhibitor for LPA₃ and LPA₁, withK_(i) values of 106 nM and 6.6 μM, respectively (Fischer et al., 2001).In order to test our high throughput test system we evaluated theeffects of DGPP 8:0 in the same stably transfected RH7777 cell lines.The K_(i) values were 202 nM for LPA₃ and 4.3 μM for LPA₁ (Table 3).These results convincingly showed the reproducibility of the DGPPresults, even after the modification of the original assay method.Ki16425 was synthesized and identified as a subtype-selective antagonistfor LPA₁ and LPA₃ with a very weak inhibitory effect on LPA₂ with K_(i)values 250 nM, 360 nM, and 5.6 μM, respectively, using GTPγS loadingassay in HEK293T cells transfected with LPA receptors (Ohta et al.,2003). When this compound was tested in our high throughputintracellular Ca²⁺ monitoring system, we obtained similar K_(i) valuesfor LPA₁ (425 nM) and LPA₃ (148 nM), however Ki16425 seemed to inhibitLPA₃ slightly better compared to LPA₁ (Table 3). N-acyl serinephosphoric acid and N-acyl tyrosine phosphoric acid were originallyidentified as inhibitors of LPA-induced platelet aggregation (Sugiura etal., 1994) and inhibitors of the LPA induced Cl⁻ current in Xenopusoocytes (Liliom et al., 1996). In a mammalian cell line, however, Ser-PAwas found to be an LPA-like agonist (Hooks et al., 1998). It was alsoshown to be an agonist at LPA₁ and LPA₂ when these receptor subtypeswere heterologously expressed in TAg-Jurkat T-cells (An et al., 1998b).In our experiments Ser-PA was a full agonist at LPA₁ (EC₅₀=1.85 μM), butonly a weak agonist at LPA₂. At LPA₃, Ser-PA was also a weak but fullagonist with an EC₅₀ value of 1.6 μM (Table 3).

An et al. (1998b) showed that Tyr-PA did not affect LPA signaling atLPA₁ and LPA₂ receptors when applied at a concentration of 1 μM. Tyr-PAin our experiments had no effect on LPA₁, however it was found to be aweak agonist at LPA₂ (EC₅₀=11 μM) and an inhibitor at LPA₃ (K_(i)=2.3μM) as shown in Table 3. VPC12249 is a 2-substituted analog of theN-acyl ethanolamide phosphate that was identified as a subtype-selectiveinhibitor of the LPA₁ and LPA₃ receptors, using a GTPγS-loading assaywith cell membranes isolated from HEK293T cells expressing LPA₁, LPA₂,or LPA₃. VPC12249 was a better antagonist at LPA₁ (K_(i)=137 nM) than atLPA₃ (K_(i)=428 nM) (Heise et al. 2001). In our experiments howeverVPC12249 was only a weak inhibitor at LPA, and a better inhibitor atLPA₃ with a K_(i) value of 588 nM (Table 3). This value is reasonablyclose to the published data in addition to the observation that VPC12249did not affect LPA signaling through LPA₂ (Table 3). Analogous to theFAPs, these compounds also showed effects that are combination ofeffects at three LPA receptors on PC-3 cells, further validating ourassay system.

In addition to its plasma membrane receptors, LPA was shown to be anagonist of the nuclear transcription factor PPARγ (McIntyre et al.,2003). Many agents have been reported to activate PPARγ, includingthiazolidinedione family represented by Rosiglitazone, oxidizedphospholipids, fatty acids, eicosanoids, and oxidized LDL. Zhang et alshowed that unsaturated and alkyl ether analogs of LPA,1,1-difluorodeoxy-(2R)-palmitoyl-sn-glycero-3-phosphate, its mono-fluoroanalog 1-palmitoyl-(2R)-fluorodeoxy-sn-glycero-3-phosphate, and theoxidized phosphatidylcholine1-O-hexadecyl-2-azeleoyl-phosphatidylcholine induced neointimaformation, an early step leading to the development of atherogenicplaques, through PPARγ activation (Zhang et al., 2004).

The SAR of neointima formation by LPA analogs in vivo was identical toPPARγ activation in vitro and different from LPA G-protein coupledreceptors (Zhang et al., 2004). We tested selected compounds includingFAP-12 (2b), unsaturated thiophosphate analogs (8d-g),tetradecylphosphonate 9c, previously reported LPA₁/LPA₃ antagonistsDGPP, Ki16425, VPC12249, and thiophosphate analog OMPT, a selective LPA₃agonist, for PPARγ activation in vitro in CV1 cells using thePPRE-Acox-Rluc reporter gene assay. Interestingly, results from thisassay (FIG. 4) indicate that along with previously reported agonists(OMPT) and antagonists (DGPP, Ki16425, VPC12249) FAP analogs, which haveLPA₁₋₃ agonist/antagonist activities, can activate PPRE-Acox-Rlucreporter. These results are consistent with previously reported results(Zhang et al., 2004) in that LPA GPCR ligands can activate PPARγ.However, the results also emphasize that the SAR of PPARγ activation isdifferent from GPCRs.

The present study extended the validity of our previously describedtwo-point contact model as the minimal requirement to elicit specificinteractions with LPA GPCRs, and provides further refinement of theminimal pharmacophore FAP by identifying modifications that allowed thesynthesis of a pan-agonist and a pan-antagonist and severalsubtype-selective ligands. A systematic SAR study of the FAPpharmacophore with phosphonate, thiophosphate and introduction ofunsaturation in the side chain outlined important principles for thedesign of subtype-selective LPA receptor agonists and antagonists. Theresults of the FAP analogs, and previously reported LPA agonists andantagonists by other groups, obtained from transfected RH7777 cellsexpressing each LPA receptor individually were consistent with resultsobtained from PC-3 cells that endogenously express LPA₁₋₃ receptors. Inaddition to their ligand properties on LPA GPCR, we showed that FAPsalso activate nuclear transcription factor PPARγ with an SAR differentfrom LPA GPCR. Based on the principles that emerged from SAR of FAP-12,oleoyl-thiophosphate (8 g) was synthesized and identified as a novelpan-agonist at all three LPA receptors confirming the previouslypredicted necessity for an LPA₁₋₃ agonist to possess both appropriatecharge and side chain (length and unsaturation). Tetradecyl-phosphonate(9c) was identified as a metabolically stable first pan-antagonist thatcould serve as a lead structure for further development of LPA₁₋₃receptor antagonists that are not sensitive to degradation by lipidphosphate phosphatases. Our results provide the first comprehensiveevaluation of LPA-GPCR ligands as agonists of PPARγ. It was anunexpected surprise that with the exception of VPC12249 all otheranalogs, regardless of their agonist or antagonist activity on LPA GPCR,were agonists of PPARγ. TABLE 3 Effects of FAP analogs 5a-f, 8a-g, 9a-dand 11 on LPA₁₋₃ transfected RH7777 cells and comparison of theactivities with the previously reported compounds

LPA₁ LPA₂ LPA₃ PC-3 EC₅₀ IC₅₀ EC₅₀ IC₅₀ EC₅₀ IC₅₀ EC₅₀ IC₅₀ (E_(max))(K_(i)) (E_(max)) (K_(i)) (E_(max)) (K_(i)) (E_(max)) (K_(i)) Cmp X Y RnM nM nM nM nM nM nM nM  2a^(b) O O —(CH₂)₉CH₃  NE^(c) NE 1800 (82) NENE  384 (121)  ND^(d) ND  2b^(b) O O —(CH₂)₁₁CH₃ NE 2800 3100 (50) NE NE 128 (61) ND ND (1354)  2c^(b) O O —(CH₂)₁₃CH₃ NE 2300 NE NE NE  422(211) ND ND (1081)  5a O O —(CH₂)₈CH═CH₂ NE >10000 3800 (100) NE NE  770(504)  NA^(e) 1510 (574)  5b O O —(CH₂)₃CH═CH(CH₂)₄CH₃ NE >10000 >10000NE NE  830 (370) NA 1300 (735)  5c O O —(CH₂)₈CH═CHCH₂CH₃ NE >10000  717(78) NE NE  32 (27) NA  916 (390)  5d O O —(CH2)₈CH═CH(CH₂)₃CH₃ NE 3000 397 (58) NE NE  96 (58) NA  241 (123) (1146)  5e O O—(CH₂)₁₀CH═CHCH₂CH₃ NE 2200 4100 (75) NE NE  103 (40) ND ND (457)  5f OO —(CH₂)₈CH═CH(CH₂)₇CH₃ NE NE NE NE NE NE —(11) NA  8a O S —(CH₂)₉CH₃ NENE 4570 (100) NE NE  122 (49) NA 1220 (521)  8b O S —(CH₂)₁₁CH₃ NE NE1000 (100) NE NE  28 (14) NA 2838 (1300)  8c O S —(CH₂)₁₃CH₃ NE NE 2500(100) NE NE  162 (76) NE NE  8d O S —(CH₂)₈CH═CH₂ NE NE >10000 (56) NENE  340 (128) NA 1000 (533)  8e O S —(CH₂)₈CH═CHCH₂CH₃ NE NE  677 (100)NE NE  27 (14) —(27) 2972 (1460)  8f O S —(CH₂)₈CH═CH(CH₂)₃CH₃ NE NE 480 (150) NE NE  28 (14) —(40)  938 (397)  8g O S —(CH₂)₈CH═CH(CH₂)₇CH₃193 NE  244 (175) NE 546 NE —(30) NA (80) (78)  9a CH₂ O —(CH₂)₈CH₃ NENE NE NE NE 1200 (68) NA 3122 (1500)  9b CH₂ O —(CH₂)₁₀CH₃ NE NE NE NENE  654 (303) NA 2638 (1270)  9c CH₂ O —(CH₂)₁₂CH₃ NE ˜10000 NE 5500 NE3100 NA 9674 (4620) (3550) (1120)  9d CH₂ O —(CH₂)₁₆CH₃ NE NE NE NE NENE NE NE 11 CF₂ O —(CH₂)₁₃CH₃ NE 2500 ˜10000 NE NE 1513 ND ND (788)^(f)(40) (575) DGPP^(g) NE 5500 NE NE NE  454 (202) ND ND (4300) Ki16425^(h)NE  762 NE NE NE  301 (148) NA 3384 (1740)  (425) Ser-PA 1850 NE >10000NE 1600 NE —(42) NA (100) (100) Tyr-PA NE NE ˜11000 NE NE 5570 —(25)WA^(i) (2325) VPC12249^(j) NE WA NE NE NE 1186 NA WA (588)^(a)E_(max) = maximal efficacy of the drug/maximal efficacy of LPA 18:1,expressed as the percentage.^(b)Previously reported in Virag et al. (2003).^(c)NE = no effect was shown at the highest concentration (30 μM)tested.^(d)ND = not determined.^(e)NA = not applicable.^(f)Partial antagonist with 40% inhibition of the LPA response.^(g)Reported Ki values of DGPP are 106 nM and 6.6 μM at LPA3 and LPA1,respectively (Hasegawa et al., 2003).^(h)Reported Ki values of Ki16425 are 250 nM, 360 nM and 5.6 μM at LPA1,LPA3 and LPA2, respectively (Virag et al., 2003).^(i)WA = weak antagonist.^(j)Reported Ki values of VPC12249 are 137 nM and 428 nM at LPA1 andLPA3, respectively (Ohta et al., 2003).

Example 8 In vitro Evaluation of Compound 8g For Protection ofIntestinal Epithelial Cells Against Radiation or Chemotherapy InducedApoptosis

The experimental procedure utilized was substantially the same as thatreported in Deng et al. (2002) and Deng et al., (2003).

Basically, IEC-6 cells were grown in DMEM medium supplemented with 5%fetal bovine serum, insulin (10 μg/ml), gentamycin sulfate (50 μg/ml),and incubated at 37° C. in a humidified 90% air-10% CO₂ atmosphere.Medium was changed every other day. Sub-confluent cells were washedtwice and replaced by DMEM without serum the night before experiments.

Damage and IEC-6 cell apoptosis was induced via either γ-irradiation orchemotherapy. 20 Gy single dose of [¹³⁷Cs] source γ-irradiation was usedin all experiments. Serum starved IEC-6 cells were pretreated with LPA,FAP12, or compound 8 g (FAP 18:1d9) for 15 minutes and then irradiatedwith a Mark I Model 25 Gamma Irradiator (J. L. Shepherd & Associate, SanFernando, Calif.) at a rate of 416 R/min for 4.81 minutes on a rotatingplatform. In some experiments, LPA was added at different times beforeor after irradiation. Treatment with 20 μM camptothecin of IEC-6 cellsinduces DNA fragmentation as measured by the ELISA assay at 16 h aftertreatment. DNA fragmentation was quantified using the Cell DeathDetection ELISA kit from Boehringer (Indianapolis, Ind.) according tothe instructions of the manufacturer. Samples were run in triplicate. Aduplicate of the sample was used to quantify protein concentration usingthe BCA kit from Pierce (Rockford, Ill.). DNA fragmentation wasexpressed as absorbance units per μg protein per minute.

LPA and FAP 12 (both 10 μM) inhibited Campthotecin-induced (20 μM) DNAfragmentation in IEC-6 cells. The effect of FAPs is dose dependent asillustrated for FAP 18:1d9 thiophosphate (8 g) in FIG. 5 and iscomparable to that of LPA but supersedes it at concentrations above 3μM.

Example 9 In vivo Evaluation of Compound 8g for Protection of IntestinalEpithelial Cells Against Radiation or Chemotherapy Induced Apoptosis

The experimental procedure utilized was substantially the same as thatreported in Deng et al. (2002).

The whole body irradiation (WBI) protocol has been reviewed and approvedby the ACUC Committee of the University of Tennessee Health SciencesCenter. ICR strain male mice (Harlan Laboratories, body weight 30-33 g)on a 12 h light/dark cycle and otherwise maintained on a standardlaboratory chow ad libitum were starved for 16 h prior to treatment. WBIwas done with a 12 Gy or 15 Gy dose using Cs¹³⁷ source at a dose rate of1.9 Gy per minute. Groups of four mice received either 250 μl of 1 mMLPA complexed with 100 μM BSA dissolved in Hanks basal salt solution orthe BSA vehicle alone 2 h prior to irradiation.

For detection of the apoptotic bodies, mice were euthanized with carbondioxide inhalation 4 h after irradiation and the small intestine wasdissected and fixed in neutral phosphate buffered isotonic 10% formalin.Four ˜3- to 4-mm long segments from the small intestine were embedded inparaffin, 5 μM thick sections were cut and stained with hematoxilin andeosin. The number of surviving crypts was counted 3.5 days afterirradiation.

FAP 18:1d9 (200 μM into the stomach 2 h prior irradiation) significantly(P>0.01) enhanced crypt survival in the irradiated animals (FIG. 6). Theeffect of FAP was dose-dependent (FIG. 7). The effect of FAP 18:1d9 waspresent in the jejunum and ileum and exceeded that of LPA (FIG. 8).

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Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A method for protecting intestinal cells in a mammalian subject fromapoptosis, the method comprising administering to the subject atherapeutically effective amount of a composition comprising a compoundof formula (I)

wherein, X¹ is R¹—Y¹-A-; X² is -Z¹-P(S)(OH)₂; X³ is hydrogen; A is adirect link or (CH₂)_(l) with l being an integer from 1-30; Y¹ is(CH₂)_(l) with l being an integer from 1-30; Z¹ is oxygen; Q¹ and Q² areindependently selected from the group consisting of H, ═NR⁴, ═O, or—NR⁵R⁶; R¹ is independently a straight or branched-chain C₁ to C₃₀alkyl, a straight or branched-chain C₂ to C₃₀ alkenyl, an aromatic orheteroaromatic ring (optionally substituted), an acyl including a C₁ toC₃₀ alkyl, an aromatic or heteroaromatic ring, an arylalkyl includingstraight or branched-chain C₁ to C₃₀ alkyl, an aryloxyalkyl includingstraight or branched-chain C₁ to C₃₀ alkyl,

R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are independently hydrogen, a straight orbranched-chain C₁ to C₃₀ alkyl, a straight or branched-chain C₂ to C₃₀alkenyl, an aromatic or heteroaromatic ring with or without mono-, di-,or tri-substitutions of the ring, an acyl including a C₁ to C₃₀ alkyl oraromatic or heteroaromatic ring, an arylalkyl including straight orbranched-chain C₁ to C₃₀ alky, or an aryloxyalkyl including straight orbranched-chain C₁ to C₃₀ alkyl.
 2. The method of claim 1 wherein Q¹ andQ² of compound (I) are hydrogen and R¹ is a straight or branched-chainC₂ to C₃₀ alkenyl.
 3. The method of claim 1 wherein the compositioncomprises thiophosphoric acid O-octadec-9-enyl ester.
 4. The method ofclaim 1 wherein the step of administering is performed via a methodchosen from among the group consisting of oral, topical, transdermal,parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal,intranasal, intracavitary, intravesicalar, intraocular, intraarterial,intralesional, or a combination thereof.
 5. A method for treatingradiation injury, the method comprising administering to a mammaliansubject a composition comprising a compound of formula (I)

wherein, X¹ is R¹—Y¹-A-; X² is -Z¹-P(S)(OH)₂; X³ is hydrogen; A is adirect link or (CH₂)_(l) with l being an integer from 1-30; Y¹ is(CH₂)_(l) with l being an integer from 1-30; Z¹ is oxygen; Q¹ and Q² areindependently selected from the group consisting of H, ═NR⁴, ═O, or—NR⁵R⁶; R¹ is independently a straight or branched-chain C₁ to C₃₀alkyl, a straight or branched-chain C₂ to C₃₀ alkenyl, an aromatic orheteroaromatic ring (optionally substituted), an acyl including a C₁ toC₃₀ alkyl, an aromatic or heteroaromatic ring, an arylalkyl includingstraight or branched-chain C₁ to C₃₀ alkyl, an aryloxyalkyl includingstraight or branched-chain C₁ to C₃₀ alkyl,

R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are independently hydrogen, a straight orbranched-chain C₁ to C₃₀ alkyl, a straight or branched-chain C₂ to C₃₀alkenyl, an aromatic or heteroaromatic ring with or without mono-, di-,or tri-substitutions of the ring, an acyl including a C₁ to C₃₀ alkyl oraromatic or heteroaromatic ring, an arylalkyl including straight orbranched-chain C₁ to C₃₀ alky, or an aryloxyalkyl including straight orbranched-chain C₁ to C₃₀ alkyl.
 6. The method of claim 5 wherein Q¹ andQ² of compound (I) are hydrogen and R¹ is a straight or branched-chainC₂ to C₃₀ alkenyl.
 7. The method of claim 5 wherein the radiation isgamma radiation.
 8. The method of claim 5 wherein the radiation iswhole-body irradiation.
 9. The method of claim 5 wherein the step ofadministering is performed via oral, parenteral, subcutaneous,intravenous, or intraperitoneal route.
 10. A method for treatingchemotherapy-induced tissue damage in the intestine of a mammaliansubject, the method comprising administering to the subject acomposition comprising a compound of formula (I)

wherein, X¹ is R¹—Y¹-A-; X² is -Z¹-P(S)(OH)₂; X³ is hydrogen; A is adirect link or (CH₂)_(l) with l being an integer from 1-30; Y¹ is(CH₂)_(l) with l being an integer from 1-30; Z¹ is oxygen; Q¹ and Q² areindependently selected from the group consisting of H, ═NR⁴, ═O, or—NR⁵R⁶; R¹ is independently a straight or branched-chain C₁ to C₃₀alkyl, a straight or branched-chain C₂ to C₃₀ alkenyl, an aromatic orheteroaromatic ring (optionally substituted), an acyl including a C₁ toC₃₀ alkyl, an aromatic or heteroaromatic ring, an arylalkyl includingstraight or branched-chain C₁ to C₃₀ alkyl, an aryloxyalkyl includingstraight or branched-chain C₁ to C₃₀ alkyl,

R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ are independently hydrogen, a straight orbranched-chain C₁ to C₃₀ alkyl, a straight or branched-chain C₂ to C₃₀alkenyl, an aromatic or heteroaromatic ring with or without mono-, di-,or tri-substitutions of the ring, an acyl including a C₁ to C₃₀ alkyl oraromatic or heteroaromatic ring, an arylalkyl including straight orbranched-chain C₁ to C₃₀ alky, or an aryloxyalkyl including straight orbranched-chain C₁ to C₃₀ alkyl.
 11. The method of claim 10 wherein Q¹and Q² of compound (I) are hydrogen and R¹ is a straight orbranched-chain C₂ to C₃₀ alkenyl.
 12. The method of claim 10 wherein thestep of administering is performed via oral, parenteral, subcutaneous,intravenous, or intraperitoneal route.